Recurrent gene fusions in prostate cancer

ABSTRACT

Recurrent gene fusions in prostate cancer of androgen regulated genes or housekeeping genes and ETS family member genes are described. Compositions and methods having utility in prostate cancer diagnosis, research, and therapy are also provided.

This application is a continuation-in-part of application Ser. No.11/825,552, filed Jul. 6, 2007, which is a continuation-in-part ofapplication Ser. No. 11/519,397, filed Sep. 12, 2006, which claimspriority to provisional patent application Ser. Nos. 60/716,436, filedSep. 12, 2005, 60/779,041, filed Mar. 3, 2006, 60/730,358, filed Oct.27, 2005, and 60/795,590, filed Apr. 28, 2006, each of which is hereinincorporated by reference in its entirety.

This invention was made with government support under CA069568 andCA111275 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for cancerdiagnosis, research and therapy, including but not limited to, cancermarkers. In particular, the present invention relates to recurrent genefusions as diagnostic markers and clinical targets for prostate cancer.

BACKGROUND OF THE INVENTION

A central aim in cancer research is to identify altered genes that arecausally implicated in oncogenesis. Several types of somatic mutationshave been identified including base substitutions, insertions,deletions, translocations, and chromosomal gains and losses, all ofwhich result in altered activity of an oncogene or tumor suppressorgene. First hypothesized in the early 1900's, there is now compellingevidence for a causal role for chromosomal rearrangements in cancer(Rowley, Nat Rev Cancer 1: 245 (2001)). Recurrent chromosomalaberrations were thought to be primarily characteristic of leukemias,lymphomas, and sarcomas. Epithelial tumors (carcinomas), which are muchmore common and contribute to a relatively large fraction of themorbidity and mortality associated with human cancer, comprise less than1% of the known, disease-specific chromosomal rearrangements (Mitelman,Mutat Res 462: 247 (2000)). While hematological malignancies are oftencharacterized by balanced, disease-specific chromosomal rearrangements,most solid tumors have a plethora of non-specific chromosomalaberrations. It is thought that the karyotypic complexity of solidtumors is due to secondary alterations acquired through cancer evolutionor progression.

Two primary mechanisms of chromosomal rearrangements have beendescribed. In one mechanism, promoter/enhancer elements of one gene arerearranged adjacent to a proto-oncogene, thus causing altered expressionof an oncogenic protein. This type of translocation is exemplified bythe apposition of immunoglobulin (IG) and T-cell receptor (TCR) genes toMYC leading to activation of this oncogene in B- and T-cellmalignancies, respectively (Rabbitts, Nature 372: 143 (1994)). In thesecond mechanism, rearrangement results in the fusion of two genes,which produces a fusion protein that may have a new function or alteredactivity. The prototypic example of this translocation is the BCR-ABLgene fusion in chronic myelogenous leukemia (CML) (Rowley, Nature 243:290 (1973); de Klein et al., Nature 300: 765 (1982)). Importantly, thisfinding led to the rational development of imatinib mesylate (Gleevec),which successfully targets the BCR-ABL kinase (Deininger et al., Blood105: 2640 (2005)). Thus, identifying recurrent gene rearrangements incommon epithelial tumors may have profound implications for cancer drugdiscovery efforts as well as patient treatment.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides a method foridentifying prostate cancer in a patient comprising: providing a samplefrom the patient; and, detecting the presence or absence in the sampleof a gene fusion having a 5′ portion from a transcriptional regulatoryregion of an SLC45A3 gene and a 3′ portion from an ERG gene, whereindetecting the presence in the sample of the gene fusion identifiesprostate cancer in the patient. In some embodiments, the transcriptionalregulatory region of the SLC45A3 gene comprises a promoter region of theSLC45A3 gene. In some embodiments, the detecting step comprisesdetecting chromosomal rearrangements of genomic DNA having a 5′ DNAportion from the transcriptional regulatory region of the SLC45A3 geneand a 3′ DNA portion from the ERG gene. In some embodiments, thedetecting step comprises detecting chimeric mRNA transcripts having a 5′RNA portion transcribed from the transcriptional regulatory region ofthe SLC45A3 gene and a 3′ RNA portion transcribed from the ERG gene. Insome embodiments, the sample is tissue, blood, plasma, serum, urine,urine supernatant, urine cell pellet, semen, prostatic secretions orprostate cells.

In some embodiments, the present invention provides a method foridentifying prostate cancer in a patient comprising: providing a samplefrom the patient; and, detecting the presence or absence in the sampleof a gene fusion having a 5′ portion from a transcriptional regulatoryregion of an FLJ35294 gene and a 3′ portion from an ETS family membergene, wherein detecting the presence in the sample of the gene fusionidentifies prostate cancer in the patient. In some embodiments, thetranscriptional regulatory region of the FLJ35294 gene comprises apromoter region of the FLJ35294 gene. In some embodiments, the ETSfamily member gene is ETV1. In some embodiments, the detecting stepcomprises detecting chromosomal rearrangements of genomic DNA having a5′ DNA portion from the transcriptional regulatory region of theFLJ35294 gene and a 3′ DNA portion from the ETS family member gene. Insome embodiments, the detecting step comprises detecting chimeric mRNAtranscripts having a 5′ RNA portion transcribed from the transcriptionalregulatory region of the FLJ35294 gene and a 3′ RNA portion transcribedfrom the the ETS family member gene. In some embodiments, the sample istissue, blood, plasma, serum, urine, urine supernatant, urine cellpellet, semen, prostatic secretions or prostate cells.

In some embodiments, the present invention provides a method foridentifying prostate cancer in a patient comprising: providing a samplefrom the patient; and, detecting the presence or absence in the sampleof a gene fusion having a 5′ portion from a DDX5 gene and a 3′ portionfrom an ETS family member gene, wherein detecting the presence in thesample of the gene fusion identifies prostate cancer in the patient. Insome embodiments, the ETS family member gene is ETV4. In someembodiments, the detecting step comprises detecting chromosomalrearrangements of genomic DNA having a 5′ DNA portion from the DDX5 geneand a 3′ DNA portion from the ETS family member gene. In someembodiments, the detecting step comprises detecting chimeric mRNAtranscripts having a 5′ RNA portion transcribed from the DDX5 gene and a3′ RNA portion transcribed from the ETS family member gene. In someembodiments, the detecting step comprises detecting a chimeric proteinhaving an amino-terminal portion encoded by the DDX5 gene and acarboxy-terminal portion encoded by the ETS family member gene. In someembodiments, the sample is tissue, blood, plasma, serum, urine, urinesupernatant, urine cell pellet, semen, prostatic secretions or prostatecells.

In some embodiments, the present invention provides compositions fordiagnostic, therapeutic and research purposes. In some embodiments, thecomposition comprises an oligonucleotide probe comprising a sequencethat hybridizes to a junction of a chimeric genomic DNA or chimericmRNA. In some embodiments, the composition comprises a firstoligonucleotide probe comprising a sequence that hybridizes to a 5′portion of a chimeric genomic DNA or chimeric mRNA and a secondoligonucleotide probe comprising a sequence that hybridizes to a 3′portion of the chimeric genomic DNA or chimeric mRNA. In someembodiments, the composition comprises a first amplificationoligonucleotide comprising a sequence that hybridizes to a 5′ portion ofa chimeric genomic DNA or chimeric mRNA and a second amplificationoligonucleotide comprising a sequence that hybridizes to a 3′ portion ofthe chimeric genomic DNA or chimeric. In some embodiments, thecomposition comprises an antibody to a chimeric protein having anamino-terminal portion encoded by the 5′ gene and a carboxy-terminalportion encoded by the 3′ gene.

Additional embodiments of the present invention are provided in thedescription and examples below.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the Cancer Outlier Profile Analysis (COPA) of microarraydata. (A) ETV1 (left panels) and ERG (middle panels) expression(normalized expression units) are shown from all profiled samples in twolarge scale gene expression studies. (B) As in (A), except data fromlaser capture microdissected samples were used. (C) As in (A), exceptoncogenes (FGFR3 and CCND1) with known translocations to theimmunoglobulin heavy chain promoter (IgH) in multiple myeloma wereexamined.

FIG. 2 shows the identification and characterization of TMPRSS2:ETV1 andTMPRSS2:ERG gene fusions in prostate cancer (PCA). (A) Prostate cancercell lines (DuCaP, LnCaP and VCaP) and hormone refractory metastatic(MET) prostate cancer tissues were analyzed for ERG (▪) and ETV1 (□)mRNA expression by quantitative PCR (QPCR). (B) Loss of over-expressionof ETV1 exons 2 and 3 in MET26 compared to LNCaP cells. (C) Schematic of5′ RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE)results for ETV1 in MET26-LN and ERG in MET28-LN revealing gene fusionswith TMPRSS2. (D) Validation of TMPRSS2:ETV1 expression usingtranslocation-specific QPCR in MET26-LN and MET26-RP. (E) Validation ofTMPRSS2:ERG expression using translocation-specific QPCR in cell linesand PCA specimens.

FIG. 3 shows interphase fluorescence in situ hybridization (FISH) onformalin-fixed paraffin embedded tissue sections that confirmsTMPRSS2:ETV1 gene fusion and ERG gene rearrangement. (A and B) showtwo-color, fusion-signal approach to detect the fusion of TMPRSS2 (greensignal) and ETV1 (red signal). (C and D) Detection of ERG generearrangements using a two-color split-signal approach with two probesspanning the 5′ (green signal) and 3′ (red signal) regions of ERG. (E)Matrix representation of FISH results using the same probes as (A-D) onan independent tissue microarray containing cores from 13 cases ofclinically localized prostate cancer (PCA) and 16 cases of metastaticprostate cancer (MET).

FIG. 4 shows androgen regulation of ERG in prostate cancer cellscarrying the TMPRSS2:ERG translocation.

FIG. 5 shows Cancer Outlier Profile Analysis (COPA). FIG. 5A shows aschematic of COPA analysis. FIG. 5B shows that RUNX1T1 (ETO) had thehighest scoring outlier profile at the 90th percentile in the Valk etal. acute myeloid leukemia dataset (n=293).

FIG. 6 shows a schematic of RNA ligase-mediated rapid amplification ofcDNA ends (RLM-RACE) results for ETV1 in MET26-LN and ERG in PCA4revealing gene fusions with TMPRSS2 (TMPRSS2:ERGb fusion).

FIG. 7 shows over-expression of ETS family members in prostate cancer.Expression of all monitored ETS family members in profiled benignprostate, prostatic intraepithelial neoplasia (PIN), clinicallylocalized prostate cancer and metastatic prostate cancer from grosslydissected tissue (A) or tissue isolated by laser capture microdissection(B) was visualized using Oncomine.

FIG. 8 shows over expression of TMPRSS2 and ETV4 loci in a prostatecancer case that over-expresses ETV4. A. Expression of the indicatedexons or region of ETV4 in pooled benign prostate tissue (CPP), prostatecancers that did not over-express ETV4 and were either TMPRSS2:ERGpositive (PCA1-2) or negative (PCA3-4), and the prostate cancer casefrom our LCM cohort with ETV4 over-overexpression (PCA5). B. RLM-RACEreveals fusion of sequences upstream of TMPRSS2 with ETV4 in PCA5. C.Expression of TMPRSS2:ETV4a and TMPRSS2:ETV4b in PCA5 by QPCR. D.Interphase fluorescence in situ hybridization on formalin-fixedparaffin-embedded tissue confirms fusion of TMPRSS2 and ETV4 loci inPCA5.

FIG. 9 shows mRNA sequences of exemplary ETS family genes.

FIG. 10 shows the mRNA sequence of TMPRSS2.

FIG. 11 shows TMPRSS2:ERG gene fusion analysis by FISH. Panel A:Ideogram, depicting a break apart assay for the indirect detection ofTMPRSS2:ERG fusion. Panel B: Interphase nuclei of a stromal cell (left)and a prostate cancer gland (right). Panel C: Interphase nuclei ofprostate cancer glands showing break apart and simultaneous deletion asindicated by loss of the telomeric probe (100× oil immersion objectivemagnification). Panel D. Magnified view of boxed area in C demonstratingtwo nuclei with break apart and loss of the telomeric probe. (60× oilimmersion objective magnification).

FIG. 12 shows genomic deletions on chromosome 21 between ERG andTMPRSS2. Panel A: Samples, including 6 cell lines, 13 xenografts and 11metastatic PCA samples, were characterized for TMPRSS2:ERG andTMPRSS2:ETV1 status (gray bars for negative and blue bar for positivestatus), by qPCR and/or by FISH. Panel B: Magnification of the greenframed box in A. Panel C: Magnification of the black framed box in A.

FIG. 13 shows TMPRSS2:ERG rearrangement in clinically localized prostatecancer and association with pathological parameters. Panel A. TheTMPRSS2:ERG rearrangement was identified in 49.2% of the primary PCAsamples and 41.2% in the hormone naive metastatic LN samples. Panel B.TMPRSS2:ERG rearranged tumors with deletions tended to be observed in ahigher percentage of PCA cases with advanced tumor stage (p=0.03).

FIG. 14 shows known genes located on 21q22-23 between ERG (centromeric)and TMPRSS2 (telomeric). Genes above the black line are oriented5′-centromeric to 3′-telomeric and genes below the black line areoriented 5′-telomeric to 3′-centromeric. In the lower half of the image,a magnification of the ERG locus is depicted with FISH probes.

FIG. 15 shows ‘heterogenous’ prostate cancer case predominantly showingTMPRSS2:ERG rearrangement with the deletion (nucleus on the right) andonly small areas showing the TMPRSS2:ERG rearrangement without thedeletion (nucleus on the left).

FIG. 16 shows meta-analysis of genes located between TMPRSS2 and ERGacross 8 published expression array datasets.

FIG. 17 shows that the FISH assay detects the characteristic deletionassociated with TMPRSS2:ERG gene fusion, which is associated withdisease progression. Panels A and B: For analyzing the ERG rearrangementon chromosome 21q22.2, a break apart probe system was applied,consisting of the Biotin-14-dCTP labeled BAC clone RP11-24A11(eventually conjugated to produce a red signal) and the Digoxigenin-dUTPlabeled BAC clone RP11-137J13 (eventually conjugated to produce a greensignal), spanning the neighboring centromeric and telomeric region ofthe ERG locus, respectively. Using this break apart probe system, anucleus without ERG rearrangement exhibits two pairs of juxtaposed redand green signals. Juxtaposed red-green signals form a yellow fusionsignal (Panel B, arrow). Panel C: In a cumulative incidence regressionmodel, TMPRSS2:ERG was evaluated as a determinant for the cumulativeincidence or metastases or prostate cancer-specific death.

FIG. 18 shows FLI1 overexpression without fusion transcript.

FIG. 19 shows induction of ERG protein expression by androgen inTMPRSS2-ERG+ cells.

FIG. 20 shows a schematic of the endogenous and fusion ERG polypeptides.

FIG. 21 shows nuclear interactors for ERG2.

FIG. 22 shows sequences for peptide antibody and aqua probe generationagainst ERG1.

FIG. 23 shows sequences for peptide antibody and aqua probe generationagainst ETV1.

FIG. 24 shows sequences for peptide antibody and aqua probe generationagainst FLI1.

FIG. 25 shows sequences for peptide antibody and aqua probe generationagainst ETV4.

FIG. 26 shows the over-expression and androgen regulation of ETV1 in theLNCaP prostate cancer cell line. FIG. 26A shows expression signature ofandrogen-regulated genes in VCaP and LNCaP prostate cancer cell lines.FIG. 26B shows confirmation of PSA induction by androgen in both VCaPand LNCaP cells by quantitative PCR (QPCR). FIG. 26C shows ETV1induction by androgen in LNCaP cells. FIG. 26D shows that ETV1 ismarkedly over-expressed in LNCaP cells.

FIG. 27 shows rearrangement of ETV1 in LNCaP cells. FIG. 27A shows aschematic of BACs used as probes for fluorescence in situ hybridization(FISH). FIG. 27B shows that RP11-124L22 and RP11-1149J13 co-localize tochromosome 7 in normal peripheral lymphocytes (NPLs). FIG. 27C showslocalization of BAC #1 and BAC #4 on metaphase spreads (top panel) andinterphase cells (bottom panel) was determined in the near tetraploidLNCaP cell line. FIG. 27D shows signal from RP11-124L22 localizes tochromosome 14 in LNCaP cells.

FIG. 28 shows that the entire ETV1 locus is inserted into chromosome 14in LNCaP cells. FIG. 28A shows a schematic of BACs used in thisexperiment. FIG. 28B shows localization of RP11-124L22 (BAC #1) andRP11-313C20 (BAC #2) on metaphase spreads (top panel) and interphasecells (bottom panel) was determined by FISH in LNCaP cells.

FIG. 29 shows siRNA knockdown of ETV1 in LnCaP.

FIG. 30 shows siRNA knockdown of ERG in VCaP.

FIG. 31 shows viral overexpression systems.

FIG. 32 shows a schematic of transgenic mice.

FIG. 33 shows detection of ERG and ETV1 transcripts in urine. FIG. 33Ashows detection of ERG and ETV1 in LNCaP (high ETV1 expression) or VCaP(high ERG and TMPRSS2:ERG expression) prostate cancer cells. FIG. 33Bshows detection of ERG and ETV1 in urine of patients suspected of havingprostate cancer.

FIG. 34 shows assays used to detect TMPRSS2:ETS gene fusions in prostatecancer. FIG. 34A shows break apart assays for TMPRSS2 and ERG. An ERGrearrangement positive case (without deletion), as indicated by one pairof split 5′ and 3′ signals, is shown in the left panel. A TMPRSS2rearrangement positive case (with deletion), as indicated by a loss ofone 3′ signal, is shown in the right panel. FIG. 34B shows a fusionassay for TMPRSS2:ETV1 gene fusions. FIG. 34C shows a break apart assayfor ETV4.

FIG. 35 shows TMPRSS2, ERG, ETV1 and ETV4 rearrangements as detected byFISH. FIG. 35A shows a Table of results for rearrangements in TMPRSS2,ERG, ETV1 and ETV4 as detected by the assays shown in FIG. 34. FIG. 34Bshows a heat map representation of the TMPRSS2, ERG, ETV1 and ETV4status from the 38 cases where all four assays were evaluable asdescribed in A. FIG. 34C shows a heat map representation of cases withdiscordant TMPRSS2 and ETS rearrangement status.

FIG. 36 shows the sequences of gene fusions of the present invention.

FIG. 37 shows primers and probes for FLI-1 expression analysis.

FIG. 38 shows identification of prostate-specific or ubiquitously activeregulatory elements fused to ETV1 in outlier tumor specimens. a.Identification of ETV1 outlier cases. b. Structure of novel 5′ partnersfused to ETV1 in outlier cases. c. FISH confirmation of ETV1 fusionsusing probes 5′ to the indicated partner and 3′ to ETV1. d. Tissuespecificity of 5′ fusion partners. e. Assessment of androgen regulationof 5′ fusion partners.

FIG. 39 shows the confirmation of novel ETV1 gene fusions by qPCR.

FIG. 40 shows confirmation of novel ETV1 gene fusions by FISH

a. Schematic of BACs located 5′ and 3′ to ETV1, HNRPA2B1,HERV-K_(—)22q11.23, C15ORF21 and SLC45A3 used as probes for interphaseFISH. b-d. FISH was performed using BACs as indicated with thecorresponding fluorescent label on formalin-fixed paraffin-embeddedtissue sections for b) split signal assay of the 5′ fusion partner, c)fusion of the 5′ partner and ETV1, and d) split signal assay of ETV1.

FIG. 41 shows that ETV1 over-expression in prostate cells confersinvasiveness.

a. Adenoviruses and lentiviruses expressing the ETV1 gene fusion product(exons 4-through the reported stop codon). b. The benign immortalizedprostate cell line RWPE was infected with ETV1 or control (GUS)lentivirus as indicated, and stable clones were generated and assayedfor invasion through a modified basement membrane. c. Primary prostaticepithelial cells (PrEC) were infected with ETV1 or LACZ adenovirus asindicated and assayed for invasion. Mean (n=3)+S.E. are shown.Photomicrographs of invaded cells are indicated in the insets. d-e.siRNA knockdown of ETV1 in LNCaP cells inhibits invasion. LNCaP cellswere treated with transfection reagent alone (Untreated), or transfectedwith non-targeting or ETV1 siRNA as indicated. d. ETV1 knockdown wasconfirmed by qPCR (Mean (n=4)+S.E.). e. Cells were assessed for invasionas in b and c (Mean (n=3)+S.E.). f. RWPE-ETV1 and RWPE-GUS cells wereprofiled on Agilent Whole Genome microarrays. Network view of themolecular concept analysis of genes over-expressed in RWPE-ETV1 comparedto RWPE-GUS cells is shown. Each node represents a molecular concept, orset of biologically related genes. The node size is proportional to thenumber of genes in the concept (as examples, the “RWPE-ETV1” and“Extracellular matrix” concepts contain 527 and 186 genes,respectively). The concept color indicates the concept type according tothe legend. Each edge represents a significant enrichment (P<0.005). g.qPCR confirmation of selected genes involved in invasion over-expressedin RWPE-ETV1 cells.

FIG. 42 shows that transgenic mice expressing an ETV1 gene fusionproduct in the prostate develop mouse prostatic intraepithelialneoplasia (mPIN). a-f. Hematoxylin and eosin staining of ARR2Pb-ETV1prostates for morphological assessment. c. High power view of b, withthe inset showing prominent nucleoli in the mPIN lesion. d. Normalglands and foci of mPIN as observed in the ventral prostate (VP) ofARR2Pb-ETV1 mouse #3 (33 weeks). e. High power view of d. f. A singlegland showing normal epithelial architecture as well as mPIN. The insetshows the foci of mPIN indicated by the arrowhead. g-1.Immunohistochemistry with smooth muscle actin (SMA) demonstrates acontinuous fibromuscular layer around g) benign glands and h) all mPINlesions, while the basal cell markers i-j) cytokeratin 5 (CK5) and k-l)p63 demonstrate loss of circumferential basal cells in mPIN foci (j,l)compared to normal glands (i,k) in the dorsolateral prostate (DLP) ofARR2Pb-ETV1 mouse #3. Original magnification for a, b & d is 100× and c& e-1 is 400×.

FIG. 43 shows that distinct classes of chromosomal rearrangementsactivate ETS oncogenes in prostate cancer.

FIG. 44 shows that over-expression of ETV1 does not effect proliferationor transform benign prostatic epithelial cells. a. The benignimmortalized prostate cell line RWPE was infected with ETV1 or control(GUS) lentivirus as indicated, and stable clones were generated andassayed for proliferation. b. Primary prostatic epithelial cells (PrEC)were infected with ETV1 or LACZ adenovirus as indicated and assayedproliferation. Mean (n=3)+S.E. are shown. Results are representative ofthree independent experiments. c. ETV1 overexpression does not increasethe percentage of RWPE cells in S phase. d. ETV1 overexpression does notenhance the anchorage independent growth of RWPE cells.

FIG. 45 shows that shRNA knockdown of ETV1 inhibits invasion in LNCaPcells. a. Control LNCaP cells, or LNCaP cells infected with lentivirusesexpressing a nontargeting or ETV1 shRNAmir, as indicated, were assessedfor invasion as in FIG. 41 e.

Mean (n=3)+S.E. are shown. b. Photomicrographs of invaded cells from a.

FIG. 46 shows that over-expression of ETV1 results in decreasedexpression of proliferation related concepts.

FIG. 47 shows the identification of invasion related genesover-expressed in RWPE-ETV1 cells. a. RWPE-ETV1 and RWPE-GUS cells wereprofiled on Agilent Whole Genome microarrays. b. Overlay map identifyinggenes present in the RWPE-ETV1 concept and at least 5 of the 14 otherconcepts in the enrichment network (indicated by number).

FIG. 48 shows confirmation of ARR2Pb-ETV1-FLAG expression in regions ofmPIN in the prostates of ARR2Pb-ETV1 mice. a. Low power view of theventral prostate (VP) of ARR2Pb-ETV1 mouse #4, with benign areas andmPIN foci indicated by yellow and black arrows, respectively. b. Highpower view of the benign gland indicated in a, showing a lack ofETV1-FLAG expression. c. High power view of the mPIN gland indicated ina, demonstrating ETV1-FLAG expression.

FIG. 49 shows androgen deprivation of LNCaP cells modulates theexpression of androgen regulated genes.

FIG. 50 shows that R1881 does not induce ETV1 expression in theandrogen-responsive VCaP cell line.

FIG. 51 shows sequences of additional gene fusions of the presentinvention.

FIG. 52 shows sequences of further gene fusions of the presentinvention.

FIG. 53 shows the identification of two prostate cancer (PCa) cases asshowing ETV5 outlier expression by QPCR. Panel B shows the structure ofthe fusion transcripts for both cases, as determined by RACE.

FIG. 54 shows a Schematic approach for the comprehensive FISH screen ofETS aberrations in prostate cancer. a) A split probe FISH approach wasutilized with ˜1 Mb probes flanking ETS breakpoints and applied to aprostate cancer TMA for screening genetic rearrangements involving ETSfamily genes. b) Possible outcomes from FISH-based screening of prostatecancer. c) If the 1 Mb split probes suggested an aberration, probestightly flanking the gene of interest (100˜200 Kb) were applied ontissue sections to confirm aberrations identified by the initialscreening. d) For cases rearranged for both ETS gene and known 5′partners, a fusion probe FISH assay was performed to confirm potentialgene fusion; in cases with only ETS gene rearrangement, RLM-RACE wasutilized to identify novel 5′ partners of ETS genes.

FIG. 55 shows a summary matrix of ETS and 5′ fusion partner aberrationsin prostate cancer.

FIG. 56 shows the identification of SLC45A3 as a novel 5′ fusion partnerfor ERG. a) Schematic of BACs located 5′ and 3′ to SLC45A3 and ERG usedas probes for interphase FISH. b) FISH was performed using BACs asindicated with the corresponding fluorescent label on formalin-fixedparaffin-embedded tissue sections for split signal assay of the SLC45A3,ERG respectively and fusion of the 5′ to SLC45A3 and 3′ to ERG.

FIG. 57 shows the identification of 5′ fusion partners FLJ35294, CANT1and DDX5 in prostate cancer. a) Schematic of FLJ35294-ETV1 (PCa_(—)54),CANT1-ETV4 (PCa_(—)46) and DDX5-ETV4 (PCa_(—)85) fusions identified by5′RACE analysis. The numbers in the boxes represent exons. The numbersabove the boxes indicate the last base of each exon. Untranslatedregions are represented in lighter shades (FLJ35294 is a predictedtranscript with an uncharacterized gene structure. b) A plot ofexpression of FLJ35294, CANT1 and DDX5 genes in prostate cancer comparedwith 28 other cancer types based on the International GenomicConsortium's expOdata set accessed in the Oncomine dataset.

FIG. 58 shows the confirmation of FLJ35294:ETV1, CANT1:ETV4 andDDX5:ETV4 fusions by FISH. a) Schematic of BACs located 5′ and 3′ toETV1, ETV4, FLJ35294 and CANT1 used as probes for interphase FISH. b-d)FISH was performed using BACs as indicated with the correspondingfluorescent label on formalin-fixed paraffin-embedded tissue sectionsfor b) split signal assay of ETV1 and ETV4, c) split signal assay of the5′ fusion partners, and d) fusion of the 5′ partners and ETV1 or ETV4.Split signals are indicated by arrows, and fused signals are indicatedby yellow arrows.

FIG. 59 shows androgen-regulation of the FLJ35294 and CANT1 genomiclocus and characterization of the DDX5:ETV4 fusion protein in prostatecancer. a) Expression of ERG, ETV1 and ETV4 was determined by QPCR inbenign prostate and prostate cancer cases positive for eitherFLJ35294-ETV1, CANT1-ETV4, DDX5-ETV4 or SLC45A3:ERG. b) LNCaP cells werehormone-deprived for 48 hours, treated with R1881 or ethanol control,and assayed for FLJ35294, CANT1 and DDX5 transcript expression by Q-PCR.c) AR enrichment values at the respective loci were assessed bychromatin immunoprecipitation (ChIP) analysis. d) Upper panel, aschematic diagram of the DDX5-ETV4 fusion protein indicated by aminoacids 1 to 102 of DDX5 fused to amino acids 65 to 484 of ETV4. Middlepanel, detection of FLAGtagged DDX5-ETV4 fusion protein by immunoblotanalysis of HEK 293 cells transiently transfected withpcDNA3.2-DDX5-ETV4 expression construct. Lower panel, detection ofendogenous DDX5-ETV4 fusion protein (57 kDa) in a tissue lysate derivedfrom the patient harboring the DDX5-ETV4 gene fusion (PCa_(—)85).

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “gene fusion” refers to a chimeric genomic DNA,a chimeric messenger RNA, a truncated protein or a chimeric proteinresulting from the fusion of at least a portion of a first gene to atleast a portion of a second gene. The gene fusion need not includeentire genes or exons of genes.

As used herein, the term “gene upregulated in cancer” refers to a genethat is expressed (e.g., mRNA or protein expression) at a higher levelin cancer (e.g., prostate cancer) relative to the level in othertissues. In some embodiments, genes upregulated in cancer are expressedat a level at least 10%, preferably at least 25%, even more preferablyat least 50%, still more preferably at least 100%, yet more preferablyat least 200%, and most preferably at least 300% higher than the levelof expression in other tissues. In some embodiments, genes upregulatedin prostate cancer are “androgen regulated genes.”

As used herein, the term “gene upregulated in prostate tissue” refers toa gene that is expressed (e.g., mRNA or protein expression) at a higherlevel in prostate tissue relative to the level in other tissue. In someembodiments, genes upregulated in prostate tissue are expressed at alevel at least 10%, preferably at least 25%, even more preferably atleast 50%, still more preferably at least 100%, yet more preferably atleast 200%, and most preferably at least 300% higher than the level ofexpression in other tissues. In some embodiments, genes upregulated inprostate tissue are exclusively expressed in prostate tissue.

As used herein, the term “high expression promoter” refers to a promoterthat when fused to a gene causes the gene to be expressed in aparticular tissue (e.g., prostate) at a higher level (e.g, at a level atleast 10%, preferably at least 25%, even more preferably at least 50%,still more preferably at least 100%, yet more preferably at least 200%,and most preferably at least 300% higher) than the level of expressionof the gene when not fused to the high expression promoter. In someembodiments, high expression promoters are promoters from an androgenregulated gene or a housekeeping gene (e.g., HNRPA2B1).

As used herein, the term “transcriptional regulatory region” refers tothe region of a gene comprising sequences that modulate (e.g.,upregulate or downregulate) expression of the gene. In some embodiments,the transcriptional regulatory region of a gene comprises non-codingupstream sequence of a gene, also called the 5′ untranslated region(5′UTR). In other embodiments, the transcriptional regulatory regioncontains sequences located within the coding region of a gene or withinan intron (e.g., enhancers).

As used herein, the term “androgen regulated gene” refers to a gene orportion of a gene whose expression is induced or repressed by anandrogen (e.g., testosterone). The promoter region of an androgenregulated gene may contain an “androgen response element” that interactswith androgens or androgen signaling molecules (e.g., downstreamsignaling molecules).

As used herein, the terms “detect”, “detecting” or “detection” maydescribe either the general act of discovering or discerning or thespecific observation of a detectably labeled composition.

As used herein, the term “inhibits at least one biological activity of agene fusion” refers to any agent that decreases any activity of a genefusion of the present invention (e.g., including, but not limited to,the activities described herein), via directly contacting gene fusionprotein, contacting gene fusion mRNA or genomic DNA, causingconformational changes of gene fusion polypeptides, decreasing genefusion protein levels, or interfering with gene fusion interactions withsignaling partners, and affecting the expression of gene fusion targetgenes. Inhibitors also include molecules that indirectly regulate genefusion biological activity by intercepting upstream signaling molecules.

As used herein, the term “siRNAs” refers to small interfering RNAs. Insome embodiments, siRNAs comprise a duplex, or double-stranded region,of about 18-25 nucleotides long; often siRNAs contain from about two tofour unpaired nucleotides at the 3′ end of each strand. At least onestrand of the duplex or double-stranded region of a siRNA issubstantially homologous to, or substantially complementary to, a targetRNA molecule. The strand complementary to a target RNA molecule is the“antisense strand;” the strand homologous to the target RNA molecule isthe “sense strand,” and is also complementary to the siRNA antisensestrand. siRNAs may also contain additional sequences; non-limitingexamples of such sequences include linking sequences, or loops, as wellas stem and other folded structures. siRNAs appear to function as keyintermediaries in triggering RNA interference in invertebrates and invertebrates, and in triggering sequence-specific RNA degradation duringposttranscriptional gene silencing in plants.

The term “RNA interference” or “RNAi” refers to the silencing ordecreasing of gene expression by siRNAs. It is the process ofsequence-specific, post-transcriptional gene silencing in animals andplants, initiated by siRNA that is homologous in its duplex region tothe sequence of the silenced gene. The gene may be endogenous orexogenous to the organism, present integrated into a chromosome orpresent in a transfection vector that is not integrated into the genome.The expression of the gene is either completely or partially inhibited.RNAi may also be considered to inhibit the function of a target RNA; thefunction of the target RNA may be complete or partial.

As used herein, the term “stage of cancer” refers to a qualitative orquantitative assessment of the level of advancement of a cancer.Criteria used to determine the stage of a cancer include, but are notlimited to, the size of the tumor and the extent of metastases (e.g.,localized or distant).

As used herein, the term “gene transfer system” refers to any means ofdelivering a composition comprising a nucleic acid sequence to a cell ortissue. For example, gene transfer systems include, but are not limitedto, vectors (e.g., retroviral, adenoviral, adeno-associated viral, andother nucleic acid-based delivery systems), microinjection of nakednucleic acid, polymer-based delivery systems (e.g., liposome-based andmetallic particle-based systems), biolistic injection, and the like. Asused herein, the term “viral gene transfer system” refers to genetransfer systems comprising viral elements (e.g., intact viruses,modified viruses and viral components such as nucleic acids or proteins)to facilitate delivery of the sample to a desired cell or tissue. Asused herein, the term “adenovirus gene transfer system” refers to genetransfer systems comprising intact or altered viruses belonging to thefamily Adenoviridae.

As used herein, the term “site-specific recombination target sequences”refers to nucleic acid sequences that provide recognition sequences forrecombination factors and the location where recombination takes place.

As used herein, the term “nucleic acid molecule” refers to any nucleicacid containing molecule, including but not limited to, DNA or RNA. Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of apolypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region of a structural gene and thesequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. Sequenceslocated 5′ of the coding region and present on the mRNA are referred toas 5′ non-translated sequences. Sequences located 3′ or downstream ofthe coding region and present on the mRNA are referred to as 3′non-translated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

As used herein, the term “heterologous gene” refers to a gene that isnot in its natural environment. For example, a heterologous geneincludes a gene from one species introduced into another species. Aheterologous gene also includes a gene native to an organism that hasbeen altered in some way (e.g., mutated, added in multiple copies,linked to non-native regulatory sequences, etc). Heterologous genes aredistinguished from endogenous genes in that the heterologous genesequences are typically joined to DNA sequences that are not foundnaturally associated with the gene sequences in the chromosome or areassociated with portions of the chromosome not found in nature (e.g.,genes expressed in loci where the gene is not normally expressed).

As used herein, the term “oligonucleotide,” refers to a short length ofsingle-stranded polynucleotide chain. Oligonucleotides are typicallyless than 200 residues long (e.g., between 15 and 100), however, as usedherein, the term is also intended to encompass longer polynucleotidechains. Oligonucleotides are often referred to by their length. Forexample a 24 residue oligonucleotide is referred to as a “24-mer”.Oligonucleotides can form secondary and tertiary structures byself-hybridizing or by hybridizing to other polynucleotides. Suchstructures can include, but are not limited to, duplexes, hairpins,cruciforms, bends, and triplexes.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, the sequence“5′-A-G-T-3′,” is complementary to the sequence “3′-T-C-A-5′.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This is of particular importance inamplification reactions, as well as detection methods that depend uponbinding between nucleic acids.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is a nucleic acid molecule that at leastpartially inhibits a completely complementary nucleic acid molecule fromhybridizing to a target nucleic acid is “substantially homologous.” Theinhibition of hybridization of the completely complementary sequence tothe target sequence may be examined using a hybridization assay(Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially homologous sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely homologous nucleic acid molecule to a target underconditions of low stringency. This is not to say that conditions of lowstringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second target that issubstantially non-complementary (e.g., less than about 30% identity); inthe absence of non-specific binding the probe will not hybridize to thesecond non-complementary target.

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described above.

A gene may produce multiple RNA species that are generated bydifferential splicing of the primary RNA transcript. cDNAs that aresplice variants of the same gene will contain regions of sequenceidentity or complete homology (representing the presence of the sameexon or portion of the same exon on both cDNAs) and regions of completenon-identity (for example, representing the presence of exon “A” on cDNA1 wherein cDNA 2 contains exon “B” instead). Because the two cDNAscontain regions of sequence identity they will both hybridize to a probederived from the entire gene or portions of the gene containingsequences found on both cDNAs; the two splice variants are thereforesubstantially homologous to such a probe and to each other.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(i.e., it is the complement of) the single-stranded nucleic acidsequence under conditions of low stringency as described above.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids. A single molecule that contains pairing of complementarynucleic acids within its structure is said to be “self-hybridized.”

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. Under “low stringency conditions” anucleic acid sequence of interest will hybridize to its exactcomplement, sequences with single base mismatches, closely relatedsequences (e.g., sequences with 90% or greater homology), and sequenceshaving only partial homology (e.g., sequences with 50-90% homology).Under ‘medium stringency conditions,” a nucleic acid sequence ofinterest will hybridize only to its exact complement, sequences withsingle base mismatches, and closely relation sequences (e.g., 90% orgreater homology). Under “high stringency conditions,” a nucleic acidsequence of interest will hybridize only to its exact complement, and(depending on conditions such a temperature) sequences with single basemismatches. In other words, under conditions of high stringency thetemperature can be raised so as to exclude hybridization to sequenceswith single base mismatches.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5×Denhardt's reagent [50×Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and100 μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 5×SSPE, 0.1% SDS at 42° C. when a probe of about 500nucleotides in length is employed.

The art knows well that numerous equivalent conditions may be employedto comprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (e.g., thepresence or absence of formamide, dextran sulfate, polyethylene glycol)are considered and the hybridization solution may be varied to generateconditions of low stringency hybridization different from, butequivalent to, the above listed conditions. In addition, the art knowsconditions that promote hybridization under conditions of highstringency (e.g., increasing the temperature of the hybridization and/orwash steps, the use of formamide in the hybridization solution, etc.)(see definition above for “stringency”).

As used herein, the term “amplification oligonucleotide” refers to anoligonucleotide that hybridizes to a target nucleic acid, or itscomplement, and participates in a nucleic acid amplification reaction.An example of an amplification oligonucleotide is a “primer” thathybridizes to a template nucleic acid and contains a 3′ OH end that isextended by a polymerase in an amplification process. Another example ofan amplification oligonucleotide is an oligonucleotide that is notextended by a polymerase (e.g., because it has a 3′ blocked end) butparticipates in or facilitates amplification. Amplificationoligonucleotides may optionally include modified nucleotides or analogs,or additional nucleotides that participate in an amplification reactionbut are not complementary to or contained in the target nucleic acid.Amplification oligonucleotides may contain a sequence that is notcomplementary to the target or template sequence. For example, the 5′region of a primer may include a promoter sequence that isnon-complementary to the target nucleic acid (referred to as a“promoter-primer”). Those skilled in the art will understand that anamplification oligonucleotide that functions as a primer may be modifiedto include a 5′ promoter sequence, and thus function as apromoter-primer. Similarly, a promoter-primer may be modified by removalof, or synthesis without, a promoter sequence and still function as aprimer. A 3′ blocked amplification oligonucleotide may provide apromoter sequence and serve as a template for polymerization (referredto as a “promoter-provider”).

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, that is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product that is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, that is capable of hybridizing to at least a portion ofanother oligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present invention will be labeled with any “reportermolecule,” so that is detectable in any detection system, including, butnot limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. It is not intended that the present invention be limited to anyparticular detection system or label.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecomponent or contaminant with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is such present in a form orsetting that is different from that in which it is found in nature. Incontrast, non-isolated nucleic acids as nucleic acids such as DNA andRNA found in the state they exist in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding a given protein includes, by way ofexample, such nucleic acid in cells ordinarily expressing the givenprotein where the nucleic acid is in a chromosomal location differentfrom that of natural cells, or is otherwise flanked by a differentnucleic acid sequence than that found in nature. The isolated nucleicacid, oligonucleotide, or polynucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acid,oligonucleotide or polynucleotide is to be utilized to express aprotein, the oligonucleotide or polynucleotide will contain at a minimumthe sense or coding strand (i.e., the oligonucleotide or polynucleotidemay be single-stranded), but may contain both the sense and anti-sensestrands (i.e., the oligonucleotide or polynucleotide may bedouble-stranded).

As used herein, the term “purified” or “to purify” refers to the removalof components (e.g., contaminants) from a sample. For example,antibodies are purified by removal of contaminating non-immunoglobulinproteins; they are also purified by the removal of immunoglobulin thatdoes not bind to the target molecule. The removal of non-immunoglobulinproteins and/or the removal of immunoglobulins that do not bind to thetarget molecule results in an increase in the percent of target-reactiveimmunoglobulins in the sample. In another example, recombinantpolypeptides are expressed in bacterial host cells and the polypeptidesare purified by the removal of host cell proteins; the percent ofrecombinant polypeptides is thereby increased in the sample.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of recurrent genefusions in prostate cancer. The present invention provides diagnostic,research, and therapeutic methods that either directly or indirectlydetect or target the gene fusions. The present invention also providescompositions for diagnostic, research, and therapeutic purposes.

I. Gene Fusions

The present invention identifies recurrent gene fusions indicative ofprostate cancer. The gene fusions are the result of a chromosomalrearrangement of an androgen regulated gene (ARG) or housekeeping gene(HG) and an ETS family member gene. Despite their recurrence, thejunction where the ARG or HG fuses to the ETS family member gene varies.The gene fusions typically comprise a 5′ portion from a transcriptionalregulatory region of an ARG or HG and a 3′ portion from an ETS familymember gene. The recurrent gene fusions have use as diagnostic markersand clinical targets for prostate cancer.

A. Androgen Regulated Genes

Genes regulated by androgenic hormones are of critical importance forthe normal physiological function of the human prostate gland. They alsocontribute to the development and progression of prostate carcinoma.Recognized ARGs include, but are not limited to: TMPRSS2; SLC45A3;HERV-K_(—)22q11.23; C15ORF21; FLJ35294; CANT1; PSA; PSMA; KLK2; SNRK;Seladin-1; and, FKBP51 (Paoloni-Giacobino et al., Genomics 44: 309(1997); Velasco et al., Endocrinology 145(8): 3913 (2004)).

TMPRSS2 (NM_(—)005656) has been demonstrated to be highly expressed inprostate epithelium relative to other normal human tissues (Lin et al.,Cancer Research 59: 4180 (1999)). The TMPRSS2 gene is located onchromosome 21. This gene is located at 41,750,797-41,801,948 bp from thepter (51,151 total bp; minus strand orientation). The human TMPRSS2protein sequence may be found at GenBank accession no. AAC51784 (SwissProtein accession no. 015393) and the corresponding cDNA at GenBankaccession no. U75329 (see also, Paoloni-Giacobino, et al., Genomics 44:309 (1997)).

SLC45A3, also known as prostein or P501S, has been shown to beexclusively expressed in normal prostate and prostate cancer at both thetranscript and protein level (Kalos et al., Prostate 60, 246-56 (2004);Xu et al., Cancer Res 61, 1563-8 (2001)).

HERV-K_(—)22q11.23, by EST analysis and massively parallel sequencing,was found to be the second most strongly expressed member of the HERV-Kfamily of human endogenous retroviral elements and was most highlyexpressed in the prostate compared to other normal tissues (Stauffer etal., Cancer Immun 4, 2 (2004)). While androgen regulation of HERV-Kelements has not been described, endogenous retroviral elements havebeen shown to confer androgen responsiveness to the mouse sex-linkedprotein gene C4A (Stavenhagen et al., Cell 55, 247-54 (1988)). OtherHERV-K family members have been shown to be both highly expressed andestrogen-regulated in breast cancer and breast cancer cell lines (Ono etal., J Virol 61, 2059-62 (1987); Patience et al., J Virol 70, 2654-7(1996); Wang-Johanning et al., Oncogene 22, 1528-35 (2003)), andsequence from a HERV-K3 element on chromosome 19 was fused to FGFR1 in acase of stem cell myeloproliferative disorder with t(8;19)(p12;q13.3)(Guasch et al., Blood 101, 286-8 (2003)).

C15ORF21, also known as D-PCA-2, was originally isolated based on itsexclusive over-expression in normal prostate and prostate cancer (Weigleet al., Int J Cancer 109, 882-92 (2004)).

FLJ35294 was identified as a member of the “full-length long Japan”(FLJ) collection of sequenced human cDNAs (Nat. Genet. 2004 January; 36(1):40-5. Epub 2003 Dec. 21).

CANT1, also known as sSCAN1, is a soluble calcium-activated nucleotidase(Arch Biochem Biophys. 2002 Oct. 1; 406 (1):105-15). CANT1 is a371-amino acid protein. A cleavable signal peptide generates a secretedprotein of 333 residues with a predicted core molecular mass of 37,193Da. Northern analysis identified the transcript in a range of humantissues, including testis, placenta, prostate, and lung. No traditionalapyrase-conserved regions or nucleotide-binding domains were identifiedin this human enzyme, indicating membership in a new family ofextracellular nucleotidases.

Gene fusions of the present invention may comprise transcriptionalregulatory regions of an ARG. The transcriptional regulatory region ofan ARG may contain coding or non-coding regions of the ARG, includingthe promoter region. The promoter region of the ARG may further comprisean androgen response element (ARE) of the ARG. The promoter region forTMPRSS2, in particular, is provided by GenBank accession numberAJ276404.

B. Housekeeping Genes

Housekeeping genes are constitutively expressed and are generallyubiquitously expressed in all tissues. These genes encode proteins thatprovide the basic, essential functions that all cells need to survive.Housekeeping genes are usually expressed at the same level in all cellsand tissues, but with some variances, especially during cell growth andorganism development. It is unknown exactly how many housekeeping geneshuman cells have, but most estimates are in the range from 300-500.

Many of the hundreds of housekeeping genes have been identified. Themost commonly known gene, GAPDH (glyceraldehyde-3-phosphatedehydrogenase), codes for an enzyme that is vital to the glycolyticpathway. Another important housekeeping gene is albumin, which assistsin transporting compounds throughout the body. Several housekeepinggenes code for structural proteins that make up the cytoskeleton such asbeta-actin and tubulin. Others code for 18S or 28S rRNA subunits of theribosome. HNRPA2B1 is a member of the ubiquitously expressedheteronuclear ribonuclear proteins. Its promoter has been shown to beunmetheylated and prevents transcriptional silencing of the CMV promoterin transgenes (Williams et al., BMC Biotechnol 5, 17 (2005)). Anexemplary listing of housekeeping genes can be found, for example, inTrends in Genetics, 19, 362-365 (2003).

C. ETS Family Member Genes

The ETS family of transcription factors regulate the intra-cellularsignaling pathways controlling gene expression. As downstream effectors,they activate or repress specific target genes. As upstream effectors,they are responsible for the spacial and temporal expression of numerousgrowth factor receptors. Almost 30 members of this family have beenidentified and implicated in a wide range of physiological andpathological processes. These include, but are not limited to: ERG; ETV1(ER81); FLI1; ETS1; ETS2; ELK1; ETV6 (TEL1); ETV7 (TEL2); GABPα; ELF1;ETV4 (E1AF; PEA3); ETV5 (ERM); ERF; PEA3/E1AF; PU.1; ESE1/ESX; SAP1(ELK4); ETV3 (METS); EWS/FLI1; ESE1; ESE2 (ELF5); ESE3; PDEF; NET (ELK3;SAP2); NERF (ELF2); and FEV. Exemplary ETS family member sequences aregiven in FIG. 9.

ERG (NM_(—)004449) has been demonstrated to be highly expressed inprostate epithelium relative to other normal human tissues. The ERG geneis located on chromosome 21. The gene is located at38,675,671-38,955,488 base pairs from the pter. The ERG gene is 279,817total bp minus strand orientation. The corresponding ERG cDNA andprotein sequences are given at GenBank accession nos. M17254 and NP04440(Swiss Protein acc. no. P11308), respectively.

The ETV1 gene is located on chromosome 7 (GenBank accession nos.NC_(—)000007.11; NC_(—)086703.11; and NT_(—)007819.15). The gene islocated at 13,708330-13,803,555 base pairs from the pter. The ETV1 geneis 95,225 bp total, minus strand orientation. The corresponding ETV1cDNA and protein sequences are given at GenBank accession nos.NM_(—)004956 and NP_(—)004947 (Swiss protein acc. no. P50549),respectively.

The human ETV4 gene is located on chromosome 14 (GenBank accession nos.NC_(—)000017.9; NT_(—)010783.14; and NT_(—)086880.1). The gene is at38,960,740-38,979,228 base pairs from the pter. The ETV4 gene is 18,488bp total, minus strand orientation. The corresponding ETV4 cDNA andprotein sequences are given at GenBank accession nos. NM_(—)001986 andNP_(—)01977 (Swiss protein acc. no. P43268), respectively.

The human ETV5 gene is located on chromosome 3 at 3q28 (NC_(—)000003.10(187309570.187246803). The corresponding ETV5 mRNA and protein sequencesare given by GenBank accession nos. NM_(—)004454 and CAG33048,respectively.

D. ETS Gene Fusions

Including the initial identification of TMPRSS2:ETS gene fusions, fiveclasses of ETS rearrangements in prostate cancer have been identified(FIG. 43). The present invention is not limited to a particularmechanism. Indeed, an understanding of the mechanism is not necessary topractice the present invention. Nonetheless, it is contemplated thatupregulated expression of ETS family members via fusion with an ARG orHG or insertion into a locus with increased expression in cancerprovides a mechanism for prostate cancers. Knowledge of the class ofrearrangement present in a particular individual allows for customizedcancer therapy.

1. Classes of Gene Rearrangements

TMPRSS2:ETS gene fusions (Class I) represent the predominant class ofETS rearrangements in prostate cancer. Rearrangements involving fusionswith untranslated regions from other prostate-specific androgen-inducedgenes (Class IIa) and endogenous retroviral elements (Class IIb), suchas SLC45A3 and HERV-K_(—)22q11.23 respectively, function similarly toTMRPSS2 in ETS rearrangements. Similar to the 5′ partners in class I andII rearrangements, C15ORF21 is markedly over-expressed in prostatecancer. However, unlike fusion partners in class I and IIrearrangements, C15ORF21 is repressed by androgen, representing a novelclass of ETS rearrangements (Class III) involving prostate-specificandrogen-repressed 5′ fusion partners. By contrast, HNRPA2B1 did notshow prostate-specific expression or androgen-responsiveness. Thus,HNRPA2B1:ETV1 represents a novel class of ETS rearrangements (Class IV)where fusions involving non-tissue specific promoter elements drive ETSexpression. In Class V rearrangements, the entire ETS gene is rearrangedto prostate-specific regions.

Men with advanced prostate cancer are commonly treated withandrogen-deprivation therapy, usually resulting in tumor regression.However the cancer almost invariably progresses with ahormone-refractory phenotype. As Class IV rearrangements (such asHNRPA2B1:ETV1) are driven by androgen insensitive promoter elements, theresults indicate that these patients may not respond to anti-androgentreatment, as these gene fusions would not be responsive toandrogen-deprivation. Anti-androgen treatment of patients with Class IIIrearrangements may increase ETS fusion expression. For example,C15ORF21:ETV1 was isolated from a patient with hormone-refractorymetastatic prostate cancer where anti-androgen treatment increasedC15ORF21:ETV1 expression. Supporting this hypothesis, androgenstarvation of LNCaP significantly decreased the expression of endogenousPSA and TMPRSS2, had no effect on HNRPA2B1, and increased the expressionof C15ORF21 (FIG. 49). This allows for customized treatment of men withprostate cancer based on the class of fusion present (e.g., the choiceof androgen blocking therapy or other alternative therapies).

Multiple classes of gene rearrangements in prostate cancer indicate amore generalized role for chromosomal rearrangements in commonepithelial cancers. For example, tissue specific promoter elements maybe fused to oncogenes in other hormone driven cancers, such as estrogenresponse elements fused to oncogenes in breast cancer. Additionally,while prostate specific fusions (Classes I-III, V) would not provide agrowth advantage and be selected for in other epithelial cancers,fusions involving strong promoters of ubiquitously expressed genes, suchas HNRPA2B1, result in the aberrant expression of oncogenes across tumortypes. In summary, this study supports a role for chromosomalrearrangements in common epithelial tumor development through a varietyof mechanisms, similar to hematological malignancies.

2. ARG/ETS Gene Fusions

As described above, the present invention provides fusions of an ARG toan ETS family member gene. Exemplary gene fusion sequences are given inFIGS. 36, 51 and 52. For TMPRSS2, ERG, ETV1 and ETV4, the GenBankreference sequence ID's are provided and the exons are aligned using theMay 2004 assembly of the UCSC Human Genome. For all identified fusions,FIG. 36 provides a complete sequence from the beginning of the TMPRSS2gene through the fusion and the stop codon of the ETS family membergene. The deposited GenBank sequence for each of the published variantsis also provided. Some TMPRSS2:ERG and TMPRSS2:ETV1 fusions aredescribed by the breakpoint exons of TMPRSS2 and the ETS family membergene. For example, TMPRSS2:ERGa, which fuses exon 1 of TMPRSS2 to exons4 through 11 of ERG, is identified as TMPRSS2:ERG(1,4).

Certain gene fusions are more common than others in prostate cancer. Thepresent invention identifies 50-80% of prostate cancers as havingrecurrent gene fusions of TMPRSS2 with ERG, ETV1, ETV4 or FLI1. Ofthose, 50-70% are TMPRSS2-ERG, 50%-60% of which result from the deletionof genetic information between the TMPRSS2 and ERG locus on chromosome21 (described in more detail below), 5-10% are TMPRSS2-ETV1, 1-2% areTMPRSS2-ETV4, and 1-2% are TMPRSS2-FLI1.

Experiments conducted during the course of development of the presentinvention indicated that certain fusion genes express fusiontranscripts, while others do not express a functional transcript(Tomlins et al., Science, 310: 644-648 (2005); Tomlins et al., CancerResearch 66: 3396-3400 (2006)).

a. ERG Gene Fusions

As described above, gene fusions comprising ERG were found to be themost common gene fusions in prostate cancer. Experiments conductedduring the course of development of the present invention identifiedsignificant genomic deletions located between TMPRSS2 and ERG onchromosome 21q22.2-3. Deletions were seen in TMPRSS2:ERG fusion positivePCA samples. The deletions appear in a consensus area but showvariability within this area. In previously published work by Paris etal. (Hum. Mol. Genet. 13: 1303-13 (2004)), CGH analysis detecteddeletions in the CTD-2103O7 BAC that is 6 kb centromeric from TMPRSS2.These deletions were observed in 12.5% (9/72) of clinically localizedPCA samples and 33% (5/15) of the metastatic PCA samples. These resultssupport the SNP array data from the current study and indicates thateither PCA deletions become more common with progression or thatdeletions are identified more often in PCA that tend to progress morerapidly. Given the striking intra-tumoral homogeneity of the TMPRSS2:ERGrearrangements, it is more likely that these molecular sub-types areassociated with different disease progression characteristics.

One hundred eighteen clinically localized PCA cases with 49.2% harboringrearrangement of ERG were evaluated. Intronic deletions were observed in60.3% of these TMPRSS2:ERG fusion positive cases. Almost all PCA sampleswith marked over expression of ERG have a rearrangement, and the overexpression occurs in about the same number of cases as therearrangement. Using Oncomine, a publicly available compendium of geneexpression data, 4 significantly down regulated genes located in thearea of the common deletion site were identified (FIG. 16).

The present invention is not limited to a particular mechanism. Indeed,an understanding of the mechanism is not necessary to practice thepresent invention. Nonetheless, the results suggest that nearly half ofall PCAs can be defined by the TMPRSS2:ERG rearrangement. The majorityof these tumors demonstrate an intronic deletion, which according to theoligonucleotide SNP array genomic analysis is variable in size. However,approximately 30-40% did not demonstrate a deletion and thus mightharbor a balanced translocation of TMRPSS2 and ERG. This variability inthe extent of the deletion may be associated with disease progression ashas been observed with CML. The current study identified significantclinical associations with tumor stage and lymph node status.TMPRSS2:ERG rearranged tumors with deletion also showed a trend towardshigher rates of PSA biochemical failure.

Additional experiments conducted during the course of development of thepresent invention explored the risk of developing metastases or prostatecancer specific death based on the presence of the TMPRSS2:ERG genefusion in a watchful waiting cohort of early prostate cancers with longterm follow-up. The frequency of the TMPRSS2:ERG gene fusion wasassessed using 92 cases.

The frequency of TMPRSS2:ERG gene fusion in this population-based cohortwas 15.2% (14/92), lower than the 50% frequency observed in twohospital-based cohorts. The present invention is not limited to aparticular mechanism. Indeed, an understanding of the mechanism is notnecessary to practice the present invention. Nonetheless, thisdifference in TMPRSS2:ERG gene fusion prostate cancers may be due toethnic and racial genetic differences. These differences may also beexplained by the lower percentage of high grade cases in this watchfulwaiting cohort as compared to the other non-population based studies.

A significant association between TMPRSS2:ERG gene fusion anddevelopment of distant metastases and prostate cancer specific death wasobserved with a cumulative incidence ratio of 3.6 (P=0.004, 95%confidence interval=1.5 to 8.9). These data suggest that TMPRSS2:ERGgene fusion prostate cancers have a more aggressive phenotype. Furtherexperiments indicated that genomic deletions in the TMPRSS2:ERG genefusion were correlated with advanced and/or metastatic prostate cancer(See e.g., Example 5).

The present invention has also demonstrated that androgen can induce theoverexpression of ERG, presumably through AREs, in aTMPRSS2-ERG-positive cell line. The present invention is not limited toa particular mechanism. Indeed, an understanding of the mechanism is notnecessary to practice the present invention. Nonetheless, collectively,the results suggest that dysregulation of ETS family activity throughAREs upstream of TMPRSS2 may drive prostate cancer development.

b. ETV1 Gene Fusions

Further studies conducted during the course of development of someembodiments of the present invention investigated the discordancebetween the frequency of ETV1 outlier expression and TMPRSS2:ETV1positive prostate cancers. The results confirmed previous studiesidentifying TMPRSS2:ERG gene fusions as the predominant mechanismdriving ERG over-expression in prostate cancer. However, in threeprostate cancers over-expressing ETV1, novel 5′ fusion partners wereidentified. The present invention is not limited to a particularmechanism. Indeed, an understanding of the mechanism is not necessary topractice the present invention. Nonetheless, the reason for thisdiscrepancy in 5′ partners for fusions involving ERG and ETV1 isunclear, however as TMPRSS2 and ERG are located approximately ˜3 MBapart on chromosome 21, their proximity may favor TMPRSS2 as the 5′partner for ERG.

Over-expression of ETV1 under androgen regulation in a transgenic mouseresulted in highly penetrant mPIN (9 of 12, 75%) in the mouse prostate.The development of carcinoma was not observed. These results support invitro studies, where ETV1 over-expression increased invasion in benignprostatic epithelial cells but was not sufficient for transformation,and previous studies indicating that in humans, ETS fusions likely occurduring the PIN to carcinoma transition or early in prostate cancerprogression (Tomlins et al., Nat Genet 39, 41-51 (2007)). The presentinvention is not limited to a particular mechanism. Indeed, anunderstanding of the mechanism is not necessary to practice the presentinvention. Nonetheless, it is contemplated that, in human prostatecancer development, ETS gene fusions occur in the context of earlierlesions, such as loss of single NKX3-1 and/or PTEN alleles (Tomlins etal., Annual Review of Pathology: Mechanisms of Disease 1, 243-271(2006)). Furthermore, the development of mPIN in ETV1 transgenic micewithout early progression to carcinoma is similar to mouse models ofthese other early events in human prostate cancer, such as NKX3-1+/− andPTEN+/− mice (Kim et al., Proc Natl Acad Sci USA 99, 2884-9 (2002);Abdulkadir et al., Mol Cell Biol 22, 1495-503 (2002); Di Cristofano etal., Nat Genet 27, 222-4 (2001)). Thus, it is contemplated that crossesbetween ARR2Pb-ETV1 mice and these mice will produce oncogene/tumorsuppressor models mimicking early events in human prostate cancerdevelopment.

In order to identify transcriptional programs regulated by ETV1 inRWPE-ETV1, expression signatures were loaded into the Molecular ConceptsMap, an analytical framework for exploring the network ofinter-relationships among a growing collection of molecular concepts, orbiologically related gene sets. In addition to being the largestcollection of gene sets for association analysis, the MCM is unique inthat computes pair-wise associations among all gene sets in thedatabase, allowing the identification and visualization of “enrichmentnetworks” of linked concepts.

This analysis showed that an in vivo signature of genes overexpressed inprostate cancers with ETV1 outlier-expression vs. cancers withoutoutlier expression of ETS genes was enriched (P=0.003) in the in vitrosignature of genes over-expressed in RWPE-ETV1 cells (FIG. 41 f),supporting the biological relevance of the in vitro model. Moregenerally, MCM analysis identified a network of molecular conceptsrelated to cell invasion that were enriched in the ETV1 over-expressedsignature, consistent with the phenotypic effects described in thisreport. For example, the signature shared enrichment with conceptsrepresenting genes over-expressed in invasive vs. superficialtransitional cell bladder cancer (Modlich et al. P=2.9E-14, Dyrskjot etal. P=1.2E-8) 29, 30 and genes over-expressed in invasive breast ductalcarcinoma vs. ductal carcinoma in situ (Schuetz et al. P=3.8E-13). Moredirectly, the signature shared enrichment with the InterPro concept ofproteins containing “Peptidase M10A and M12B, matrixin or adamalysindomains” (P=8.5E-5), which includes matrix metalloproteinases (MMPs) anda disintegrin and metalloproteinase domains (ADAMs).

Several MMPs and members of the urokinase plasminogen activator pathway,both of which are known to mediate invasion and are reported to bedirect targets of ETS transcription factors, were over-expressed inRWPE-ETV1 cells (FIG. 41 g). Furthermore, the ‘over-expressed inRWPE-ETV1’ signature shared overlap with a signature of genesover-expressed in MCF-10A cells over-expressing the STAT3-C oncogene(P=8.9E-14). In this model, STAT3-C over-expression did not affectproliferation, but increased invasion in an MMP9 dependent manner. Theresults also parallel studies of EWSR1:ETS gene fusions in Ewing'ssarcoma, which demonstrated that knockdown or over-expression ofEWS:FLI1 affected invasion, but not proliferation (Smith et al., CancerCell 9, 405-16 (2006)). MCM analysis of the under-expressed in RWPE-ETV1signature revealed enrichment with proliferation related concepts (FIG.50), indicating subtle effects on cellular proliferation not apparent inFACS or proliferation assays.

The present study also demonstrates the utility of using outlier geneexpression to guide cytogenetic studies. For example, despite numerouskaryotyping, SKY and high resolution array CGH studies, the cryptic ETV1rearrangement has not been reported in LNCaP (Beheshti et al., Mol Diagn5, 23-32 (2000); Beheshti et al., Neoplasia 3, 62-9 (2001); Gibas etal., Cancer Genet Cytogenet 11, 399-404 (1984); Watson et al., Hum Genet120, 795-805 (2007); Pang et al., Prostate 66, 157-72 (2006); Murillo etal., Genes Chromosomes Cancer 45, 702-16 (2006); Shi et al., Prostate60, 257-71 (2004); Takaha et al., Cancer Res 62, 647-51 (2002);Strefford et al., Cancer Genet Cytogenet 124, 112-21 (2001); Thalmann etal., Cancer Res 54, 2577-81 (1994)), the most commonly used in vitromodel of prostate cancer. Similarly, TMPRSS2:ERG fusions are oftencaused by intrachromosomal deletions between TMPRSS2 and ERG on 21q,which cannot be detected by karyotyping. Likewise, the HNRPA2B1:ETV1fusion identified here also occurs through an intrachromosomal deletionspanning approximately 15 MB. These results indicate that crypticrearrangements and intrachromosomal deletions that activate oncogenesmay occur in other common epithelial carcinomas.

c. ETV4 Gene Fusions

Experiments were conducted to interrogate the expression of all ETSfamily members monitored in prostate cancer profiling studies from theOncomine database (Rhodes et al., supra). Marked over-expression of theETS family member ETV4 was identified in a single prostate cancer casefrom each of two studies—one profiling grossly dissected tissues(Lapointe et al., supra) (FIG. 7A) and the other profiling laser capturemicrodissected (LCM) tissues1 (FIG. 7B). ETV4 was found to be fused toTMPRSS2 in cancerous tissues.

d. ETV5 Gene Fusions

Additional experiments identified TMPRSS2:ETV5 and SLC45A3:ETV5 genefusions (Example 20). ETV5 was found to have outlier expression inprostate cancer sample and was subsequently found to be present in genefusions.

3. HG/ETS Gene Fusions

Additional experiments identified HNRPA2B1:ETV1 gene fusions in prostatecancer. HNRPA2B1 did not show prostate-specific expression or androgenregulation, and is instead expressed strongly across all tumor types.HNRPA2B1 encodes a member of the ubiquitously expressed heteronuclearribonuclear proteins, and its promoter shares structural similarity toother housekeeping genes (Antoniou et al., Genomics 82, 269-79 (2003)).Furthermore, the HNRPA2B1 promoter has been shown to be unmetheylatedand prevents transcriptional silencing of the CMV promoter in transgenes(Williams et al., BMC Biotechnol 5, 17 (2005)). Thus, HNRPA2B1:ETV1represents a novel class of gene fusions in common epithelialcarcinomas, where non-tissue specific promoter elements drive theexpression of oncogenes. While TMPRSS2:ETS fusions are functionallyanalogous to IGH-MYC rearrangements in B cell malignancies,HNRPA2B1:ETV1 is more analogous to inv(3)(q21q26) and t(3;3)(q21;q26) inacute myeloid leukemia, which are thought to result in the placement ofEVI under the control of enhancer elements of the constitutivelyexpressed RPN1 gene (Suzukawa et al., Blood 84, 2681-8 (1994); Wieser etal., Leuk Lymphoma 43, 59-65 (2002)).

It is contemplated that other common epithelial cancers are driven bytissue specific promoter elements, such as estrogen response elementsfused to oncogenes in breast cancer. Furthermore, while the prostatespecific fusions (such as SLC45A3:ETV1) identified here would notprovide a growth advantage in other epithelial cancers, it iscontemplated that fusions involving strong promoters of ubiquitouslyexpressed genes, such as HNRPA2B1, can result in the aberrant expressionof oncogenes across tumor types. The HNRPA2B1:ETV1 fusion identifiedhere occurs through an intrachromosomal deletion spanning approximately15 MB. These results indicate that cryptic rearrangements andintrachromosomal deletions that activate oncogenes may occur in othercommon epithelial carcinomas.

II. Antibodies

The gene fusion proteins of the present invention, including fragments,derivatives and analogs thereof, may be used as immunogens to produceantibodies having use in the diagnostic, research, and therapeuticmethods described below. The antibodies may be polyclonal or monoclonal,chimeric, humanized, single chain or Fab fragments. Various proceduresknown to those of ordinary skill in the art may be used for theproduction and labeling of such antibodies and fragments. See, e.g.,Burns, ed., Immunochemical Protocols, 3^(rd) ed., Humana Press (2005);Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory (1988); Kozbor et al., Immunology Today 4: 72 (1983); Köhlerand Milstein, Nature 256: 495 (1975). Antibodies or fragments exploitingthe differences between the truncated ETS family member protein orchimeric protein and their respective native proteins are particularlypreferred.

III. Diagnostic Applications

The fusion of an ARG or HG to an ETS family member gene is detectable asDNA, RNA or protein. Initially, the gene fusion is detectable as achromosomal rearrangement of genomic DNA having a 5′ portion from atranscriptional regulatory region of the ARG or HG and a 3′ portion fromthe ETS family member gene. Once transcribed, the gene fusion isdetectable as a chimeric mRNA having a 5′ portion from thetranscriptional regulatory region of the ARG or HG and a 3′ portion fromthe ETS family member gene. Once translated, the gene fusion isdetectable as an amino-terminally truncated ETS family member proteinresulting from the fusion of the transcriptional regulatory region ofthe ARG or HG to the ETS family member gene; a chimeric protein havingan amino-terminal portion from the transcriptional regulatory region ofthe ARG or HG and a carboxy-terminal portion from the ETS family membergene; or, an upregulated, but otherwise indistinguishable, native ETSfamily member protein. The truncated ETS family member protein andchimeric protein may differ from their respective native proteins inamino acid sequence, post-translational processing and/or secondary,tertiary or quaternary structure. Such differences, if present, can beused to identify the presence of the gene fusion. Specific methods ofdetection are described in more detail below.

The present invention provides DNA, RNA and protein based diagnosticmethods that either directly or indirectly detect the gene fusions. Thepresent invention also provides compositions and kits for diagnosticpurposes.

The diagnostic methods of the present invention may be qualitative orquantitative. Quantitative diagnostic methods may be used, for example,to discriminate between indolent and aggressive cancers via a cutoff orthreshold level. Where applicable, qualitative or quantitativediagnostic methods may also include amplification of target, signal orintermediary (e.g., a universal primer).

An initial assay may confirm the presence of a gene fusion but notidentify the specific fusion. A secondary assay is then performed todetermine the identity of the particular fusion, if desired. The secondassay may use a different detection technology than the initial assay.

The gene fusions of the present invention may be detected along withother markers in a multiplex or panel format. Markers are selected fortheir predictive value alone or in combination with the gene fusions.Exemplary prostate cancer markers include, but are not limited to:AMACR/P504S (U.S. Pat. No. 6,262,245); PCA3 (U.S. Pat. No. 7,008,765);PCGEM1 (U.S. Pat. No. 6,828,429); prostein/P501S, P503S, P504S, P509S,P510S, prostase/P703P, P710P (U.S. Publication No. 20030185830); and,those disclosed in U.S. Pat. Nos. 5,854,206 and 6,034,218, and U.S.Publication No. 20030175736, each of which is herein incorporated byreference in its entirety. Markers for other cancers, diseases,infections, and metabolic conditions are also contemplated for inclusionin a multiplex of panel format.

The diagnostic methods of the present invention may also be modifiedwith reference to data correlating particular gene fusions with thestage, aggressiveness or progression of the disease or the presence orrisk of metastasis. Ultimately, the information provided by the methodsof the present invention will assist a physician in choosing the bestcourse of treatment for a particular patient.

A. Sample

Any patient sample suspected of containing the gene fusions may betested according to the methods of the present invention. By way ofnon-limiting examples, the sample may be tissue (e.g., a prostate biopsysample or a tissue sample obtained by prostatectomy), blood, urine,semen, prostatic secretions or a fraction thereof (e.g., plasma, serum,urine supernatant, urine cell pellet or prostate cells). A urine sampleis preferably collected immediately following an attentive digitalrectal examination (DRE), which causes prostate cells from the prostategland to shed into the urinary tract.

The patient sample typically requires preliminary processing designed toisolate or enrich the sample for the gene fusions or cells that containthe gene fusions. A variety of techniques known to those of ordinaryskill in the art may be used for this purpose, including but notlimited: centrifugation; immunocapture; cell lysis; and, nucleic acidtarget capture (See, e.g., EP Pat. No. 1 409 727, herein incorporated byreference in its entirety).

B. DNA and RNA Detection

The gene fusions of the present invention may be detected as chromosomalrearrangements of genomic DNA or chimeric mRNA using a variety ofnucleic acid techniques known to those of ordinary skill in the art,including but not limited to: nucleic acid sequencing; nucleic acidhybridization; and, nucleic acid amplification.

1. Sequencing

Illustrative non-limiting examples of nucleic acid sequencing techniquesinclude, but are not limited to, chain terminator (Sanger) sequencingand dye terminator sequencing. Those of ordinary skill in the art willrecognize that because RNA is less stable in the cell and more prone tonuclease attack experimentally RNA is usually reverse transcribed to DNAbefore sequencing.

Chain terminator sequencing uses sequence-specific termination of a DNAsynthesis reaction using modified nucleotide substrates. Extension isinitiated at a specific site on the template DNA by using a shortradioactive, or other labeled, oligonucleotide primer complementary tothe template at that region. The oligonucleotide primer is extendedusing a DNA polymerase, standard four deoxynucleotide bases, and a lowconcentration of one chain terminating nucleotide, most commonly adi-deoxynucleotide. This reaction is repeated in four separate tubeswith each of the bases taking turns as the di-deoxynucleotide. Limitedincorporation of the chain terminating nucleotide by the DNA polymeraseresults in a series of related DNA fragments that are terminated only atpositions where that particular di-deoxynucleotide is used. For eachreaction tube, the fragments are size-separated by electrophoresis in aslab polyacrylamide gel or a capillary tube filled with a viscouspolymer. The sequence is determined by reading which lane produces avisualized mark from the labeled primer as you scan from the top of thegel to the bottom.

Dye terminator sequencing alternatively labels the terminators. Completesequencing can be performed in a single reaction by labeling each of thedi-deoxynucleotide chain-terminators with a separate fluorescent dye,which fluoresces at a different wavelength.

2. Hybridization

Illustrative non-limiting examples of nucleic acid hybridizationtechniques include, but are not limited to, in situ hybridization (ISH),microarray, and Southern or Northern blot.

In situ hybridization (ISH) is a type of hybridization that uses alabeled complementary DNA or RNA strand as a probe to localize aspecific DNA or RNA sequence in a portion or section of tissue (insitu), or, if the tissue is small enough, the entire tissue (whole mountISH). DNA ISH can be used to determine the structure of chromosomes. RNAISH is used to measure and localize mRNAs and other transcripts withintissue sections or whole mounts. Sample cells and tissues are usuallytreated to fix the target transcripts in place and to increase access ofthe probe. The probe hybridizes to the target sequence at elevatedtemperature, and then the excess probe is washed away. The probe thatwas labeled with either radio-, fluorescent- or antigen-labeled bases islocalized and quantitated in the tissue using either autoradiography,fluorescence microscopy or immunohistochemistry, respectively. ISH canalso use two or more probes, labeled with radioactivity or the othernon-radioactive labels, to simultaneously detect two or moretranscripts.

a. FISH

In some embodiments, fusion sequences are detected using fluorescence insitu hybridization (FISH). The preferred FISH assays for the presentinvention utilize bacterial artificial chromosomes (BACs). These havebeen used extensively in the human genome sequencing project (see Nature409: 953-958 (2001)) and clones containing specific BACs are availablethrough distributors that can be located through many sources, e.g.,NCBI. Each BAC clone from the human genome has been given a referencename that unambiguously identifies it. These names can be used to find acorresponding GenBank sequence and to order copies of the clone from adistributor.

In some embodiments, the detection assay is a FISH assay utilizing aprobe for ETV1 (e.g., bac RP11-692L4), a set of probes for c-ERG:t-ERGbreak apart (e.g., bac RP11-24A11 and as a probe for t-ERG RP11-372O17or RP11-137J13). In other embodiments, the FISH assay is performed bytesting for ETV1 deletion or amplification with a set of probes, whereinone probe spans the ETV1 locus (e.g., bac RP11-692L4) and the otherprobe hybridizes to chromosome 7 (e.g., a probe on the centromere of thechromosome). In still further embodiments, the method is performed bytesting for ERG deletion or amplification with a set of probes, onespanning the ERG locus (e.g., bac RP11-476D17) and one reference probeon chromosome 21 (e.g., PR11-32L6; RP11-752M23; RP11-1107H21; RP11-639A7or RP11-1077M21). In yet other embodiments, the method is performed bytesting for TMPRSS2 deletion/amplification with a set of probes, onespanning the TMPRSS2 (e.g., RP11-121A5; RP11-120C17; PR11-814F13; orRR11-535H11) locus and one reference probe on chromosome 21 (e.g.,PR11-32L6; RP11-752M23; RP11-1107H21; RP11-639A7 or RP11-1077M21). Insome embodiments, the method further comprises a hybridization using aprobe selected from the group including, but not limited to RP11-121A5;RP11-120C17; PR11-814F13; and RR11-535H11.

The present invention further provides a method of performing a FISHassay on human prostate cells, human prostate tissue or on the fluidsurrounding said human prostate cells or human prostate tissue. In someembodiments, the assay comprises a hybridization step utilizing a probeselected from the group including, but not limited to, RP11-372O17;RP11-137J13; RP11-692L4; RP11-476D17; PR11-32L6; RP11-752M23;RP11-1107H21; RP11-639A7; RP11-1077M21; RP11-121A5; RP11-120C17;PR11-814F13; and RR11-535H11.

Specific BAC clones that can be used in FISH protocols to detectrearrangements relevant to the present invention are as follows:

-   -   For testing for an ETV1-TMPRSS2 fusion, one probe spanning the        ETV1 and one spanning the TMPRSS2 locus may be used:    -   BAC for ETV1: RP11-692L4    -   BAC for TMPRSS2: RP11-121A5, (RP11-120C17, PR11-814F13,        RR11-535H11)    -   Testing ERG translocation with set of probes for c-ERG:t-ERG        break apart:    -   BAC for c-ERG: RP11-24A11    -   BACs for t-ERG: RP11-372O17, RP11-137J13    -   Testing ETV1 deletion/amplification with set of probes, one        spanning the ETV1 locus and one reference probe on chromosome 7:    -   BAC for ETV1: RP11-692L4

Testing ERG deletion/amplification with set of probes, one spanning theERG locus and one reference probe on chromosome 21:

-   -   BAC for ERG: RP11-476D17    -   BACs for reference probe on chromosome 21:*    -   Testing TMPRSS2 deletion/amplification with set of probes, one        spanning the TMPRSS2 locus and one reference probe on chromosome        21:    -   BACs for TMPRSS2: RP11-121A5, (RP11-120C17, PR11-814F13,        RR11-535H11)    -   BACs for reference probe on chromosome 21: PR11-32L6,        RP11-752M23, RP11-1107H21, RP11-639A7, (RP11-1077M21).

The most preferred probes for detecting a deletion mutation resulting ina fusion between TMPRSS2 and ERG are RP11-24A11 and RP11-137J13. Theseprobes, or those described above, are labeled with appropriatefluorescent or other markers and then used in hybridizations. TheExamples section provided herein sets forth one particular protocol thatis effective for measuring deletions but one of skill in the art willrecognize that many variations of this assay can be used equally well.Specific protocols are well known in the art and can be readily adaptedfor the present invention. Guidance regarding methodology may beobtained from many references including: In situ Hybridization: MedicalApplications (eds. G. R. Coulton and J. de Belleroche), Kluwer AcademicPublishers, Boston (1992); In situ Hybridization: In Neurobiology;Advances in Methodology (eds. J. H. Eberwine, K. L. Valentino, and J. D.Barchas), Oxford University Press Inc., England (1994); In situHybridization: A Practical Approach (ed. D. G. Wilkinson), OxfordUniversity Press Inc., England (1992)); Kuo, et al., Am. J. Hum. Genet.49:112-119 (1991); Klinger, et al., Am. J. Hum. Genet. 51:55-65 (1992);and Ward, et al, Am. J. Hum. Genet. 52:854-865 (1993)). There are alsokits that are commercially available and that provide protocols forperforming FISH assays (available from e.g., Oncor, Inc., Gaithersburg,Md.). Patents providing guidance on methodology include U.S. Pat. Nos.5,225,326; 5,545,524; 6,121,489 and 6,573,043. All of these referencesare hereby incorporated by reference in their entirety and may be usedalong with similar references in the art and with the informationprovided in the Examples section herein to establish procedural stepsconvenient for a particular laboratory.

Table 13 below shows additional BAC clones that find use as FISH probes.

TABLE 13 Gene Chromosome RefSeq 5′ BAC 3′ BAC Paired EHF 11p13 NM_012153RP5-1135K18 RP5-1002E13 2 ELF1 13q14 NM_172373 RP11-88n4 RP11-53f19 ELF24q28 NM_201999.1 RP11-22o8 RP11-375P1 ELF3 1q32 NM_004433 RP11-25B7RP11-246J15 ELF4 Xq25 NM_001421 RP5-875H3 RP4-753P9 ELF5 11p13NM_001422.2 RP5-1002E13 RP5-1135K18 2 ELK1 Xp11 NM_005229 RP1-54B20RP1-306D1 ELK3 12q22 NM_005230 RP11-69E3 RP11-510I5 ELK4 1q32NM_001973.2 RP11-131E5 RP11-249h15 ERF 19q13 NM_006494.1 RP11-208I3RP11-317E13 ERG 21q22 NM_004449.3 RP11-137J13 RP11-24A11 1 ETS1 11q24NM_005238.2 RP11-254C5 RP11-112m22 ETS2 21q22 NM_005239.4 RP11-24A11RP11-137J13 1 ETV1 7p21 NM_004956.3 RP11-1149J13 RP11-34C22 ETV2 19q13NM_014209.1 RP11-32h17 RP11-92j4 ETV3 1q23 NM_005240.1 RP11-91G5RP11-1038N13 3 ETV4 17q21 NM_001986.1 RP11-436J4 RP11-100E5 ETV5 3q27NM_004454.1 RP11-379C23 RP11-1144N13 ETV6 12p13 NM_001987.3 RP11-90N7RP11-59h1 ETV7 6p21 NM_016135.2 RP3-431A14 RP1-179N16 FEV 2q35NM_017521.2 RP11-316O14 RP11-129D2 FLI1 11q24 NM_002017.2 RP11-112M22RP11-75P14 FLJ16478 1q23 NM_001004341 RP11-91G5 RP11-1038N13 3 SPDEF6p21 NM_012391.1 RP11-79j23 RP11-119c22 SPI1 11p11 NM_016135.2RP11-56e13 RP11-29o22 SPIB 19q13 NM_003121.2 RP11-510I16 RP11-26P14 SPIC12q23 NM_152323.1 RP11-426H24 RP11-938C1 TMPRSS2 21q22 NM_005656.2RP11-35C4 RP11-120C17

Additional FISH probes useful in the methods of the present inventionare shown in Table 16 (FIG. 46).

b. Microarrays

Different kinds of biological assays are called microarrays including,but not limited to: DNA microarrays (e.g., cDNA microarrays andoligonucleotide microarrays); protein microarrays; tissue microarrays;transfection or cell microarrays; chemical compound microarrays; and,antibody microarrays. A DNA microarray, commonly known as gene chip, DNAchip, or biochip, is a collection of microscopic DNA spots attached to asolid surface (e.g., glass, plastic or silicon chip) forming an arrayfor the purpose of expression profiling or monitoring expression levelsfor thousands of genes simultaneously. The affixed DNA segments areknown as probes, thousands of which can be used in a single DNAmicroarray. Microarrays can be used to identify disease genes bycomparing gene expression in disease and normal cells. Microarrays canbe fabricated using a variety of technologies, including but notlimiting: printing with fine-pointed pins onto glass slides;photolithography using pre-made masks; photolithography using dynamicmicromirror devices; ink-jet printing; or, electrochemistry onmicroelectrode arrays.

Southern and Northern blotting is used to detect specific DNA or RNAsequences, respectively. DNA or RNA extracted from a sample isfragmented, electrophoretically separated on a matrix gel, andtransferred to a membrane filter. The filter bound DNA or RNA is subjectto hybridization with a labeled probe complementary to the sequence ofinterest. Hybridized probe bound to the filter is detected. A variant ofthe procedure is the reverse Northern blot, in which the substratenucleic acid that is affixed to the membrane is a collection of isolatedDNA fragments and the probe is RNA extracted from a tissue and labeled.

3. Amplification

Chromosomal rearrangements of genomic DNA and chimeric mRNA may beamplified prior to or simultaneous with detection. Illustrativenon-limiting examples of nucleic acid amplification techniques include,but are not limited to, polymerase chain reaction (PCR), reversetranscription polymerase chain reaction (RT-PCR), transcription-mediatedamplification (TMA), ligase chain reaction (LCR), strand displacementamplification (SDA), and nucleic acid sequence based amplification(NASBA). Those of ordinary skill in the art will recognize that certainamplification techniques (e.g., PCR) require that RNA be reversedtranscribed to DNA prior to amplification (e.g., RT-PCR), whereas otheramplification techniques directly amplify RNA (e.g., TMA and NASBA).

The polymerase chain reaction (U.S. Pat. Nos. 4,683,195, 4,683,202,4,800,159 and 4,965,188, each of which is herein incorporated byreference in its entirety), commonly referred to as PCR, uses multiplecycles of denaturation, annealing of primer pairs to opposite strands,and primer extension to exponentially increase copy numbers of a targetnucleic acid sequence. In a variation called RT-PCR, reversetranscriptase (RT) is used to make a complementary DNA (cDNA) from mRNA,and the cDNA is then amplified by PCR to produce multiple copies of DNA.For other various permutations of PCR see, e.g., U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159; Mullis et al., Meth. Enzymol. 155:335 (1987); and, Murakawa et al., DNA 7: 287 (1988), each of which isherein incorporated by reference in its entirety.

Transcription mediated amplification (U.S. Pat. Nos. 5,480,784 and5,399,491, each of which is herein incorporated by reference in itsentirety), commonly referred to as TMA, synthesizes multiple copies of atarget nucleic acid sequence autocatalytically under conditions ofsubstantially constant temperature, ionic strength, and pH in whichmultiple RNA copies of the target sequence autocatalytically generateadditional copies. See, e.g., U.S. Pat. Nos. 5,399,491 and 5,824,518,each of which is herein incorporated by reference in its entirety. In avariation described in U.S. Publ. No. 20060046265 (herein incorporatedby reference in its entirety), TMA optionally incorporates the use ofblocking moieties, terminating moieties, and other modifying moieties toimprove TMA process sensitivity and accuracy.

The ligase chain reaction (Weiss, R., Science 254: 1292 (1991), hereinincorporated by reference in its entirety), commonly referred to as LCR,uses two sets of complementary DNA oligonucleotides that hybridize toadjacent regions of the target nucleic acid. The DNA oligonucleotidesare covalently linked by a DNA ligase in repeated cycles of thermaldenaturation, hybridization and ligation to produce a detectabledouble-stranded ligated oligonucleotide product.

Strand displacement amplification (Walker, G. et al., Proc. Natl. Acad.Sci. USA 89: 392-396 (1992); U.S. Pat. Nos. 5,270,184 and 5,455,166,each of which is herein incorporated by reference in its entirety),commonly referred to as SDA, uses cycles of annealing pairs of primersequences to opposite strands of a target sequence, primer extension inthe presence of a dNTPαS to produce a duplex hemiphosphorothioatedprimer extension product, endonuclease-mediated nicking of ahemimodified restriction endonuclease recognition site, andpolymerase-mediated primer extension from the 3′ end of the nick todisplace an existing strand and produce a strand for the next round ofprimer annealing, nicking and strand displacement, resulting ingeometric amplification of product. Thermophilic SDA (tSDA) usesthermophilic endonucleases and polymerases at higher temperatures inessentially the same method (EP Pat. No. 0 684 315).

Other amplification methods include, for example: nucleic acid sequencebased amplification (U.S. Pat. No. 5,130,238, herein incorporated byreference in its entirety), commonly referred to as NASBA; one that usesan RNA replicase to amplify the probe molecule itself (Lizardi et al.,BioTechnol. 6: 1197 (1988), herein incorporated by reference in itsentirety), commonly referred to as Qβ replicase; a transcription basedamplification method (Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173(1989)); and, self-sustained sequence replication (Guatelli et al.,Proc. Natl. Acad. Sci. USA 87: 1874 (1990), each of which is hereinincorporated by reference in its entirety). For further discussion ofknown amplification methods see Persing, David H., “In Vitro NucleicAcid Amplification Techniques” in Diagnostic Medical Microbiology:Principles and Applications (Persing et al., Eds.), pp. 51-87 (AmericanSociety for Microbiology, Washington, D.C. (1993)).

4. Detection Methods

Non-amplified or amplified gene fusion nucleic acids can be detected byany conventional means. For example, the gene fusions can be detected byhybridization with a detectably labeled probe and measurement of theresulting hybrids. Illustrative non-limiting examples of detectionmethods are described below.

One illustrative detection method, the Hybridization Protection Assay(HPA) involves hybridizing a chemiluminescent oligonucleotide probe(e.g., an acridinium ester-labeled (AE) probe) to the target sequence,selectively hydrolyzing the chemiluminescent label present onunhybridized probe, and measuring the chemiluminescence produced fromthe remaining probe in a luminometer. See, e.g., U.S. Pat. No. 5,283,174and Norman C. Nelson et al., Nonisotopic Probing, Blotting, andSequencing, ch. 17 (Larry J. Kricka ed., 2d ed. 1995, each of which isherein incorporated by reference in its entirety).

Another illustrative detection method provides for quantitativeevaluation of the amplification process in real-time. Evaluation of anamplification process in “real-time” involves determining the amount ofamplicon in the reaction mixture either continuously or periodicallyduring the amplification reaction, and using the determined values tocalculate the amount of target sequence initially present in the sample.A variety of methods for determining the amount of initial targetsequence present in a sample based on real-time amplification are wellknown in the art. These include methods disclosed in U.S. Pat. Nos.6,303,305 and 6,541,205, each of which is herein incorporated byreference in its entirety. Another method for determining the quantityof target sequence initially present in a sample, but which is not basedon a real-time amplification, is disclosed in U.S. Pat. No. 5,710,029,herein incorporated by reference in its entirety.

Amplification products may be detected in real-time through the use ofvarious self-hybridizing probes, most of which have a stem-loopstructure. Such self-hybridizing probes are labeled so that they emitdifferently detectable signals, depending on whether the probes are in aself-hybridized state or an altered state through hybridization to atarget sequence. By way of non-limiting example, “molecular torches” area type of self-hybridizing probe that includes distinct regions ofself-complementarity (referred to as “the target binding domain” and“the target closing domain”) which are connected by a joining region(e.g., non-nucleotide linker) and which hybridize to each other underpredetermined hybridization assay conditions. In a preferred embodiment,molecular torches contain single-stranded base regions in the targetbinding domain that are from 1 to about 20 bases in length and areaccessible for hybridization to a target sequence present in anamplification reaction under strand displacement conditions. Understrand displacement conditions, hybridization of the two complementaryregions, which may be fully or partially complementary, of the moleculartorch is favored, except in the presence of the target sequence, whichwill bind to the single-stranded region present in the target bindingdomain and displace all or a portion of the target closing domain. Thetarget binding domain and the target closing domain of a molecular torchinclude a detectable label or a pair of interacting labels (e.g.,luminescent/quencher) positioned so that a different signal is producedwhen the molecular torch is self-hybridized than when the moleculartorch is hybridized to the target sequence, thereby permitting detectionof probe:target duplexes in a test sample in the presence ofunhybridized molecular torches. Molecular torches and a variety of typesof interacting label pairs are disclosed in U.S. Pat. No. 6,534,274,herein incorporated by reference in its entirety.

Another example of a detection probe having self-complementarity is a“molecular beacon.” Molecular beacons include nucleic acid moleculeshaving a target complementary sequence, an affinity pair (or nucleicacid arms) holding the probe in a closed conformation in the absence ofa target sequence present in an amplification reaction, and a label pairthat interacts when the probe is in a closed conformation. Hybridizationof the target sequence and the target complementary sequence separatesthe members of the affinity pair, thereby shifting the probe to an openconformation. The shift to the open conformation is detectable due toreduced interaction of the label pair, which may be, for example, afluorophore and a quencher (e.g., DABCYL and EDANS). Molecular beaconsare disclosed in U.S. Pat. Nos. 5,925,517 and 6,150,097, hereinincorporated by reference in its entirety.

Other self-hybridizing probes are well known to those of ordinary skillin the art. By way of non-limiting example, probe binding pairs havinginteracting labels, such as those disclosed in U.S. Pat. No. 5,928,862(herein incorporated by reference in its entirety) might be adapted foruse in the present invention. Probe systems used to detect singlenucleotide polymorphisms (SNPs) might also be utilized in the presentinvention. Additional detection systems include “molecular switches,” asdisclosed in U.S. Publ. No. 20050042638, herein incorporated byreference in its entirety. Other probes, such as those comprisingintercalating dyes and/or fluorochromes, are also useful for detectionof amplification products in the present invention. See, e.g., U.S. Pat.No. 5,814,447 (herein incorporated by reference in its entirety).

C. Protein Detection

The gene fusions of the present invention may be detected as truncatedETS family member proteins or chimeric proteins using a variety ofprotein techniques known to those of ordinary skill in the art,including but not limited to: protein sequencing; and, immunoassays.

1. Sequencing

Illustrative non-limiting examples of protein sequencing techniquesinclude, but are not limited to, mass spectrometry and Edmandegradation.

Mass spectrometry can, in principle, sequence any size protein butbecomes computationally more difficult as size increases. A protein isdigested by an endoprotease, and the resulting solution is passedthrough a high pressure liquid chromatography column. At the end of thiscolumn, the solution is sprayed out of a narrow nozzle charged to a highpositive potential into the mass spectrometer. The charge on thedroplets causes them to fragment until only single ions remain. Thepeptides are then fragmented and the mass-charge ratios of the fragmentsmeasured. The mass spectrum is analyzed by computer and often comparedagainst a database of previously sequenced proteins in order todetermine the sequences of the fragments. The process is then repeatedwith a different digestion enzyme, and the overlaps in sequences areused to construct a sequence for the protein.

In the Edman degradation reaction, the peptide to be sequenced isadsorbed onto a solid surface (e.g., a glass fiber coated withpolybrene). The Edman reagent, phenylisothiocyanate (PTC), is added tothe adsorbed peptide, together with a mildly basic buffer solution of12% trimethylamine, and reacts with the amine group of the N-terminalamino acid. The terminal amino acid derivative can then be selectivelydetached by the addition of anhydrous acid. The derivative isomerizes togive a substituted phenylthiohydantoin, which can be washed off andidentified by chromatography, and the cycle can be repeated. Theefficiency of each step is about 98%, which allows about 50 amino acidsto be reliably determined.

2. Immunoassays

Illustrative non-limiting examples of immunoassays include, but are notlimited to: immunoprecipitation; Western blot; ELISA;immunohistochemistry; immunocytochemistry; flow cytometry; and,immuno-PCR. Polyclonal or monoclonal antibodies detectably labeled usingvarious techniques known to those of ordinary skill in the art (e.g.,calorimetric, fluorescent, chemiluminescent or radioactive) are suitablefor use in the immunoassays.

Immunoprecipitation is the technique of precipitating an antigen out ofsolution using an antibody specific to that antigen. The process can beused to identify protein complexes present in cell extracts by targetinga protein believed to be in the complex. The complexes are brought outof solution by insoluble antibody-binding proteins isolated initiallyfrom bacteria, such as Protein A and Protein G. The antibodies can alsobe coupled to sepharose beads that can easily be isolated out ofsolution. After washing, the precipitate can be analyzed using massspectrometry, Western blotting, or any number of other methods foridentifying constituents in the complex.

A Western blot, or immunoblot, is a method to detect protein in a givensample of tissue homogenate or extract. It uses gel electrophoresis toseparate denatured proteins by mass. The proteins are then transferredout of the gel and onto a membrane, typically polyvinyldiflroride ornitrocellulose, where they are probed using antibodies specific to theprotein of interest. As a result, researchers can examine the amount ofprotein in a given sample and compare levels between several groups.

An ELISA, short for Enzyme-Linked ImmunoSorbent Assay, is a biochemicaltechnique to detect the presence of an antibody or an antigen in asample. It utilizes a minimum of two antibodies, one of which isspecific to the antigen and the other of which is coupled to an enzyme.The second antibody will cause a chromogenic or fluorogenic substrate toproduce a signal. Variations of ELISA include sandwich ELISA,competitive ELISA, and ELISPOT. Because the ELISA can be performed toevaluate either the presence of antigen or the presence of antibody in asample, it is a useful tool both for determining serum antibodyconcentrations and also for detecting the presence of antigen.

Immunohistochemistry and immunocytochemistry refer to the process oflocalizing proteins in a tissue section or cell, respectively, via theprinciple of antigens in tissue or cells binding to their respectiveantibodies. Visualization is enabled by tagging the antibody with colorproducing or fluorescent tags. Typical examples of color tags include,but are not limited to, horseradish peroxidase and alkaline phosphatase.Typical examples of fluorophore tags include, but are not limited to,fluorescein isothiocyanate (FITC) or phycoerythrin (PE).

Flow cytometry is a technique for counting, examining and sortingmicroscopic particles suspended in a stream of fluid. It allowssimultaneous multiparametric analysis of the physical and/or chemicalcharacteristics of single cells flowing through an optical/electronicdetection apparatus. A beam of light (e.g., a laser) of a singlefrequency or color is directed onto a hydrodynamically focused stream offluid. A number of detectors are aimed at the point where the streampasses through the light beam; one in line with the light beam (ForwardScatter or FSC) and several perpendicular to it (Side Scatter (SSC) andone or more fluorescent detectors). Each suspended particle passingthrough the beam scatters the light in some way, and fluorescentchemicals in the particle may be excited into emitting light at a lowerfrequency than the light source. The combination of scattered andfluorescent light is picked up by the detectors, and by analyzingfluctuations in brightness at each detector, one for each fluorescentemission peak, it is possible to deduce various facts about the physicaland chemical structure of each individual particle. FSC correlates withthe cell volume and SSC correlates with the density or inner complexityof the particle (e.g., shape of the nucleus, the amount and type ofcytoplasmic granules or the membrane roughness).

Immuno-polymerase chain reaction (IPCR) utilizes nucleic acidamplification techniques to increase signal generation in antibody-basedimmunoassays. Because no protein equivalence of PCR exists, that is,proteins cannot be replicated in the same manner that nucleic acid isreplicated during PCR, the only way to increase detection sensitivity isby signal amplification. The target proteins are bound to antibodieswhich are directly or indirectly conjugated to oligonucleotides. Unboundantibodies are washed away and the remaining bound antibodies have theiroligonucleotides amplified. Protein detection occurs via detection ofamplified oligonucleotides using standard nucleic acid detectionmethods, including real-time methods.

D. Data Analysis

In some embodiments, a computer-based analysis program is used totranslate the raw data generated by the detection assay (e.g., thepresence, absence, or amount of a given gene fusion or other markers)into data of predictive value for a clinician. The clinician can accessthe predictive data using any suitable means. Thus, in some preferredembodiments, the present invention provides the further benefit that theclinician, who is not likely to be trained in genetics or molecularbiology, need not understand the raw data. The data is presenteddirectly to the clinician in its most useful form. The clinician is thenable to immediately utilize the information in order to optimize thecare of the subject.

The present invention contemplates any method capable of receiving,processing, and transmitting the information to and from laboratoriesconducting the assays, information provides, medical personal, andsubjects. For example, in some embodiments of the present invention, asample (e.g., a biopsy or a serum or urine sample) is obtained from asubject and submitted to a profiling service (e.g., clinical lab at amedical facility, genomic profiling business, etc.), located in any partof the world (e.g., in a country different than the country where thesubject resides or where the information is ultimately used) to generateraw data. Where the sample comprises a tissue or other biologicalsample, the subject may visit a medical center to have the sampleobtained and sent to the profiling center, or subjects may collect thesample themselves (e.g., a urine sample) and directly send it to aprofiling center. Where the sample comprises previously determinedbiological information, the information may be directly sent to theprofiling service by the subject (e.g., an information card containingthe information may be scanned by a computer and the data transmitted toa computer of the profiling center using an electronic communicationsystems). Once received by the profiling service, the sample isprocessed and a profile is produced (i.e., expression data), specificfor the diagnostic or prognostic information desired for the subject.

The profile data is then prepared in a format suitable forinterpretation by a treating clinician. For example, rather thanproviding raw expression data, the prepared format may represent adiagnosis or risk assessment (e.g., likelihood of cancer being present)for the subject, along with recommendations for particular treatmentoptions. The data may be displayed to the clinician by any suitablemethod. For example, in some embodiments, the profiling servicegenerates a report that can be printed for the clinician (e.g., at thepoint of care) or displayed to the clinician on a computer monitor.

In some embodiments, the information is first analyzed at the point ofcare or at a regional facility. The raw data is then sent to a centralprocessing facility for further analysis and/or to convert the raw datato information useful for a clinician or patient. The central processingfacility provides the advantage of privacy (all data is stored in acentral facility with uniform security protocols), speed, and uniformityof data analysis. The central processing facility can then control thefate of the data following treatment of the subject. For example, usingan electronic communication system, the central facility can providedata to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the datausing the electronic communication system. The subject may chose furtherintervention or counseling based on the results. In some embodiments,the data is used for research use. For example, the data may be used tofurther optimize the inclusion or elimination of markers as usefulindicators of a particular condition or stage of disease.

E. In Vivo Imaging

The gene fusions of the present invention may also be detected using invivo imaging techniques, including but not limited to: radionuclideimaging; positron emission tomography (PET); computerized axialtomography, X-ray or magnetic resonance imaging method, fluorescencedetection, and chemiluminescent detection. In some embodiments, in vivoimaging techniques are used to visualize the presence of or expressionof cancer markers in an animal (e.g., a human or non-human mammal). Forexample, in some embodiments, cancer marker mRNA or protein is labeledusing a labeled antibody specific for the cancer marker. A specificallybound and labeled antibody can be detected in an individual using an invivo imaging method, including, but not limited to, radionuclideimaging, positron emission tomography, computerized axial tomography,X-ray or magnetic resonance imaging method, fluorescence detection, andchemiluminescent detection. Methods for generating antibodies to thecancer markers of the present invention are described below.

The in vivo imaging methods of the present invention are useful in thediagnosis of cancers that express the cancer markers of the presentinvention (e.g., prostate cancer). In vivo imaging is used to visualizethe presence of a marker indicative of the cancer. Such techniques allowfor diagnosis without the use of an unpleasant biopsy. The in vivoimaging methods of the present invention are also useful for providingprognoses to cancer patients. For example, the presence of a markerindicative of cancers likely to metastasize can be detected. The in vivoimaging methods of the present invention can further be used to detectmetastatic cancers in other parts of the body.

In some embodiments, reagents (e.g., antibodies) specific for the cancermarkers of the present invention are fluorescently labeled. The labeledantibodies are introduced into a subject (e.g., orally or parenterally).Fluorescently labeled antibodies are detected using any suitable method(e.g., using the apparatus described in U.S. Pat. No. 6,198,107, hereinincorporated by reference).

In other embodiments, antibodies are radioactively labeled. The use ofantibodies for in vivo diagnosis is well known in the art. Sumerdon etal., (Nucl. Med. Biol 17:247-254 [1990] have described an optimizedantibody-chelator for the radioimmunoscintographic imaging of tumorsusing Indium-111 as the label. Griffin et al., (J Clin One 9:631-640[1991]) have described the use of this agent in detecting tumors inpatients suspected of having recurrent colorectal cancer. The use ofsimilar agents with paramagnetic ions as labels for magnetic resonanceimaging is known in the art (Lauffer, Magnetic Resonance in Medicine22:339-342 [1991]). The label used will depend on the imaging modalitychosen. Radioactive labels such as Indium-111, Technetium-99m, orIodine-131 can be used for planar scans or single photon emissioncomputed tomography (SPECT). Positron emitting labels such asFluorine-19 can also be used for positron emission tomography (PET). ForMRI, paramagnetic ions such as Gadolinium (III) or Manganese (II) can beused.

Radioactive metals with half-lives ranging from 1 hour to 3.5 days areavailable for conjugation to antibodies, such as scandium-47 (3.5 days)gallium-67 (2.8 days), gallium-68 (68 minutes), technetiium-99m (6hours), and indium-111 (3.2 days), of which gallium-67, technetium-99m,and indium-111 are preferable for gamma camera imaging, gallium-68 ispreferable for positron emission tomography.

A useful method of labeling antibodies with such radiometals is by meansof a bifunctional chelating agent, such as diethylenetriaminepentaaceticacid (DTPA), as described, for example, by Khaw et al. (Science 209:295[1980]) for In-111 and Tc-99m, and by Scheinberg et al. (Science215:1511 [1982]). Other chelating agents may also be used, but the1-(p-carboxymethoxybenzyl)EDTA and the carboxycarbonic anhydride of DTPAare advantageous because their use permits conjugation without affectingthe antibody's immunoreactivity substantially.

Another method for coupling DPTA to proteins is by use of the cyclicanhydride of DTPA, as described by Hnatowich et al. (Int. J. Appl.Radiat. Isot. 33:327 [1982]) for labeling of albumin with In-111, butwhich can be adapted for labeling of antibodies. A suitable method oflabeling antibodies with Tc-99m which does not use chelation with DPTAis the pretinning method of Crockford et al., (U.S. Pat. No. 4,323,546,herein incorporated by reference).

A preferred method of labeling immunoglobulins with Tc-99m is thatdescribed by Wong et al. (Int. J. Appl. Radiat. Isot., 29:251 [1978])for plasma protein, and recently applied successfully by Wong et al. (J.Nucl. Med., 23:229 [1981]) for labeling antibodies.

In the case of the radiometals conjugated to the specific antibody, itis likewise desirable to introduce as high a proportion of theradiolabel as possible into the antibody molecule without destroying itsimmunospecificity. A further improvement may be achieved by effectingradiolabeling in the presence of the specific cancer marker of thepresent invention, to insure that the antigen binding site on theantibody will be protected. The antigen is separated after labeling.

In still further embodiments, in vivo biophotonic imaging (Xenogen,Almeda, Calif.) is utilized for in vivo imaging. This real-time in vivoimaging utilizes luciferase. The luciferase gene is incorporated intocells, microorganisms, and animals (e.g., as a fusion protein with acancer marker of the present invention). When active, it leads to areaction that emits light. A CCD camera and software is used to capturethe image and analyze it.

F. Compositions & Kits

Compositions for use in the diagnostic methods of the present inventioninclude, but are not limited to, probes, amplification oligonucleotides,and antibodies. Particularly preferred compositions detect a productonly when an ARG or HG fuses to an ETS family member gene. Thesecompositions include: a single labeled probe comprising a sequence thathybridizes to the junction at which a 5′ portion from a transcriptionalregulatory region of an ARG or HG fuses to a 3′ portion from an ETSfamily member gene (i.e., spans the gene fusion junction); a pair ofamplification oligonucleotides wherein the first amplificationoligonucleotide comprises a sequence that hybridizes to atranscriptional regulatory region of an ARG or HG and the secondamplification oligonucleotide comprises a sequence that hybridizes to anETS family member gene; an antibody to an amino-terminally truncated ETSfamily member protein resulting from a fusion of a transcriptionalregulatory region of an ARG or HG to an ETS family member gene; or, anantibody to a chimeric protein having an amino-terminal portion from atranscriptional regulatory region of an ARG or HG and a carboxy-terminalportion from an ETS family member gene. Other useful compositions,however, include: a pair of labeled probes wherein the first labeledprobe comprises a sequence that hybridizes to a transcriptionalregulatory region of an ARG or HG and the second labeled probe comprisesa sequence that hybridizes to an ETS family member gene.

Any of these compositions, alone or in combination with othercompositions of the present invention, may be provided in the form of akit. For example, the single labeled probe and pair of amplificationoligonucleotides may be provided in a kit for the amplification anddetection of gene fusions of the present invention. Kits may furthercomprise appropriate controls and/or detection reagents. The probe andantibody compositions of the present invention may also be provided inthe form of an array.

IV. Prognostic Applications

Experiments conducted during the course of development of the presentinvention demonstrated a close correlation between gene fusions of thepresent invention and the prognosis of patients with prostate cancer(see e.g., Example 5 below). Especially in cases where a fusion resultsfrom a deletion of the genomic DNA lying between TMPRSS2 and ERG, it hasbeen found that cancer cells assume a more aggressive phenotype. Thus,in some embodiments, assays that are capable of detecting gene fusionsbetween TMPRSS2 and ERG in which there has been a deletion ofintervening DNA are used to provide prognoses and help physicians decideon an appropriate therapeutic strategy. For example, in someembodiments, patients with tumors having this particular rearrangementare treated more intensively since their prognosis is significantlyworse than patients that lack the rearrangement.

Any assay may be used to determine whether cells are present having arearrangement of the type discussed above (e.g., those described above).

Although the present invention will most preferably be used inconnection with obtaining a prognosis for prostate cancer patients,other epithelial cell tumors may also be examined and the assays andprobes described herein may be used in determining whether cancerouscells from these tumors have rearrangements that are likely to make themparticularly aggressive, i.e., likely to be invasive and metastatic.Examples of tumors that may be characterized using this procedureinclude tumors of the breast, lung, colon, ovary, uterus, esophagus,stomach, liver, kidney, brain, skin and muscle. The assays will also beof value to researchers studying these cancers in cell lines and animalmodels.

Further experiments conducted during the course of development of thepresent invention demonstrated that chromosomal deletions can bedetected by assaying samples to determine whether there is a loss ofexpression of one or more genes located in the deleted region. Forexample, approximately 2.8 megabases of genomic DNA is typically deletedin forming a fusion between TMPRSS2 and ERG and at least four geneslying in this area are lost when this occurs. These are the ETS2 gene,the WRB gene, the PCP4 gene and the MX1 gene. A decrease in one or moreof these in cancerous prostate cells suggests a poor prognosis.

Accordingly, in some embodiments, the present invention provides amethod of assaying epithelial cells for the deletion of chromosomal DNAindicative of a cancer-associated rearrangement, comprising performing aFISH assay using at least a first and a second probe, wherein the firstprobe is at least 15 nucleotides in length (e.g., at least 15, 20, 35,etc.); is bound to a first fluorescent label; and hybridizes understringent conditions to a first sequence in the human genome wherein thefirst sequence includes at least a portion of either an androgenresponsive gene (e.g., the TMPRSS2 gene) or a ETS family gene (e.g., theERG gene, the ETV1 gene, or the ETV4 gene); and the second probe: is atleast 15 nucleotides in length; is bound to a second fluorescent labelthat is different from the first fluorescent label; and hybridizes understringent conditions to a second sequence in the human genome that isdifferent from the first sequence and which includes at least a portionof an androgen responsive gene (e.g., the TMPRSS2 gene) or a ETS familygene (e.g., the ERG gene, the ETV1 gene, or the ETV4 gene).

In further embodiments, the present invention provides a method forassaying epithelial cells (e.g., prostate cells) for a deletion ofgenomic DNA indicative of a cancer-associated rearrangement, comprising:obtaining a test sample of epithelial cells; assaying the sample ofepithelial cells to determine the level of expression of one or moregenes selected from the group including, but not limited to, ETS2; WRB;PCP4; and MX1; comparing the expression level determined in step b) withthe level in a control sample; and concluding that a deletion hasoccurred if the level of expression determined for the gene in the testsample is lower than that for a control sample.

V. Drug Screening Applications

In some embodiments, the present invention provides drug screeningassays (e.g., to screen for anticancer drugs). The screening methods ofthe present invention utilize cancer markers identified using themethods of the present invention (e.g., including but not limited to,gene fusions of the present invention). For example, in someembodiments, the present invention provides methods of screening forcompounds that alter (e.g., decrease) the expression of gene fusions.The compounds or agents may interfere with transcription, byinteracting, for example, with the promoter region. The compounds oragents may interfere with mRNA produced from the fusion (e.g., by RNAinterference, antisense technologies, etc.). The compounds or agents mayinterfere with pathways that are upstream or downstream of thebiological activity of the fusion. In some embodiments, candidatecompounds are antisense or interfering RNA agents (e.g.,oligonucleotides) directed against cancer markers. In other embodiments,candidate compounds are antibodies or small molecules that specificallybind to a cancer marker regulator or expression products of the presentinvention and inhibit its biological function.

In one screening method, candidate compounds are evaluated for theirability to alter cancer marker expression by contacting a compound witha cell expressing a cancer marker and then assaying for the effect ofthe candidate compounds on expression. In some embodiments, the effectof candidate compounds on expression of a cancer marker gene is assayedfor by detecting the level of cancer marker mRNA expressed by the cell.mRNA expression can be detected by any suitable method.

In other embodiments, the effect of candidate compounds on expression ofcancer marker genes is assayed by measuring the level of polypeptideencoded by the cancer markers. The level of polypeptide expressed can bemeasured using any suitable method, including but not limited to, thosedisclosed herein.

Specifically, the present invention provides screening methods foridentifying modulators, i.e., candidate or test compounds or agents(e.g., proteins, peptides, peptidomimetics, peptoids, small molecules orother drugs) which bind to cancer markers of the present invention, havean inhibitory (or stimulatory) effect on, for example, cancer markerexpression or cancer marker activity, or have a stimulatory orinhibitory effect on, for example, the expression or activity of acancer marker substrate. Compounds thus identified can be used tomodulate the activity of target gene products (e.g., cancer markergenes) either directly or indirectly in a therapeutic protocol, toelaborate the biological function of the target gene product, or toidentify compounds that disrupt normal target gene interactions.Compounds that inhibit the activity or expression of cancer markers areuseful in the treatment of proliferative disorders, e.g., cancer,particularly prostate cancer.

In one embodiment, the invention provides assays for screening candidateor test compounds that are substrates of a cancer marker protein orpolypeptide or a biologically active portion thereof. In anotherembodiment, the invention provides assays for screening candidate ortest compounds that bind to or modulate the activity of a cancer markerprotein or polypeptide or a biologically active portion thereof.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone, which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckennann et al., J.Med. Chem. 37: 2678-85 [1994]); spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are preferred for use withpeptide libraries, while the other four approaches are applicable topeptide, non-peptide oligomer or small molecule libraries of compounds(Lam (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci.U.S.A. 90:6909 [1993]; Erb et al., Proc. Nad. Acad. Sci. USA 91:11422[1994]; Zuckermann et al., J. Med. Chem. 37:2678 [1994]; Cho et al.,Science 261:1303 [1993]; Carrell et al., Angew. Chem. Int. Ed. Engl.33.2059 [1994]; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061[1994]; and Gallop et al., J. Med. Chem. 37:1233 [1994].

Libraries of compounds may be presented in solution (e.g., Houghten,Biotechniques 13:412-421 [1992]), or on beads (Lam, Nature 354:82-84[1991]), chips (Fodor, Nature 364:555-556 [1993]), bacteria or spores(U.S. Pat. No. 5,223,409; herein incorporated by reference), plasmids(Cull et al., Proc. Nad. Acad. Sci. USA 89:18651869 [1992]) or on phage(Scott and Smith, Science 249:386-390 [1990]; Devlin Science 249:404-406[1990]; Cwirla et al., Proc. Natl. Acad. Sci. 87:6378-6382 [1990];Felici, J. Mol. Biol. 222:301 [1991]).

In one embodiment, an assay is a cell-based assay in which a cell thatexpresses a cancer marker mRNA or protein or biologically active portionthereof is contacted with a test compound, and the ability of the testcompound to the modulate cancer marker's activity is determined.Determining the ability of the test compound to modulate cancer markeractivity can be accomplished by monitoring, for example, changes inenzymatic activity, destruction or mRNA, or the like.

The ability of the test compound to modulate cancer marker binding to acompound, e.g., a cancer marker substrate or modulator, can also beevaluated. This can be accomplished, for example, by coupling thecompound, e.g., the substrate, with a radioisotope or enzymatic labelsuch that binding of the compound, e.g., the substrate, to a cancermarker can be determined by detecting the labeled compound, e.g.,substrate, in a complex.

Alternatively, the cancer marker is coupled with a radioisotope orenzymatic label to monitor the ability of a test compound to modulatecancer marker binding to a cancer marker substrate in a complex. Forexample, compounds (e.g., substrates) can be labeled with ¹²⁵I, ³⁵S ¹⁴Cor ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemmission or by scintillation counting.Alternatively, compounds can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product.

The ability of a compound (e.g., a cancer marker substrate) to interactwith a cancer marker with or without the labeling of any of theinteractants can be evaluated. For example, a microphysiorneter can beused to detect the interaction of a compound with a cancer markerwithout the labeling of either the compound or the cancer marker(McConnell et al. Science 257:1906-1912

). As used herein, a “microphysiometer” (e.g., Cytosensor) is ananalytical instrument that measures the rate at which a cell acidifiesits environment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between a compound and cancer markers.

In yet another embodiment, a cell-free assay is provided in which acancer marker protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tobind to the cancer marker protein, mRNA, or biologically active portionthereof is evaluated. Preferred biologically active portions of thecancer marker proteins or mRNA to be used in assays of the presentinvention include fragments that participate in interactions withsubstrates or other proteins, e.g., fragments with high surfaceprobability scores.

Cell-free assays involve preparing a reaction mixture of the target geneprotein and the test compound under conditions and for a time sufficientto allow the two components to interact and bind, thus forming a complexthat can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FRET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos et al., U.S. Pat. No.4,968,103; each of which is herein incorporated by reference). Afluorophore label is selected such that a first donor molecule's emittedfluorescent energy will be absorbed by a fluorescent label on a second,‘acceptor’ molecule, which in turn is able to fluoresce due to theabsorbed energy.

Alternately, the ‘donor’ protein molecule may simply utilize the naturalfluorescent energy of tryptophan residues. Labels are chosen that emitdifferent wavelengths of light, such that the ‘acceptor’ molecule labelmay be differentiated from that of the ‘donor’. Since the efficiency ofenergy transfer between the labels is related to the distance separatingthe molecules, the spatial relationship between the molecules can beassessed. In a situation in which binding occurs between the molecules,the fluorescent emission of the ‘acceptor’ molecule label should bemaximal. A FRET binding event can be conveniently measured throughstandard fluorometric detection means well known in the art (e.g., usinga fluorimeter).

In another embodiment, determining the ability of the cancer markerprotein or mRNA to bind to a target molecule can be accomplished usingreal-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolanderand Urbaniczky, Anal. Chem. 63:2338-2345 [1991] and Szabo et al. Curr.Opin. Struct. Biol. 5:699-705 [1995]). “Surface plasmon resonance” or“BIA” detects biospecific interactions in real time, without labelingany of the interactants (e.g., BIAcore). Changes in the mass at thebinding surface (indicative of a binding event) result in alterations ofthe refractive index of light near the surface (the optical phenomenonof surface plasmon resonance (SPR)), resulting in a detectable signalthat can be used as an indication of real-time reactions betweenbiological molecules.

In one embodiment, the target gene product or the test substance isanchored onto a solid phase. The target gene product/test compoundcomplexes anchored on the solid phase can be detected at the end of thereaction. Preferably, the target gene product can be anchored onto asolid surface, and the test compound, (which is not anchored), can belabeled, either directly or indirectly, with detectable labels discussedherein.

It may be desirable to immobilize cancer markers, an anti-cancer markerantibody or its target molecule to facilitate separation of complexedfrom non-complexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound to acancer marker protein, or interaction of a cancer marker protein with atarget molecule in the presence and absence of a candidate compound, canbe accomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, glutathione-S-transferase-cancermarker fusion proteins or glutathione-S-transferase/target fusionproteins can be adsorbed onto glutathione Sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione-derivatized microtiter plates,which are then combined with the test compound or the test compound andeither the non-adsorbed target protein or cancer marker protein, and themixture incubated under conditions conducive for complex formation(e.g., at physiological conditions for salt and pH). Followingincubation, the beads or microtiter plate wells are washed to remove anyunbound components, the matrix immobilized in the case of beads, complexdetermined either directly or indirectly, for example, as describedabove.

Alternatively, the complexes can be dissociated from the matrix, and thelevel of cancer markers binding or activity determined using standardtechniques. Other techniques for immobilizing either cancer markersprotein or a target molecule on matrices include using conjugation ofbiotin and streptavidin. Biotinylated cancer marker protein or targetmolecules can be prepared from biotin-NHS (N-hydroxy-succinimide) usingtechniques known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, EL), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynon-immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the immobilized component (theantibody, in turn, can be directly labeled or indirectly labeled with,e.g., a labeled anti-IgG antibody).

This assay is performed utilizing antibodies reactive with cancer markerprotein or target molecules but which do not interfere with binding ofthe cancer markers protein to its target molecule. Such antibodies canbe derivatized to the wells of the plate, and unbound target or cancermarkers protein trapped in the wells by antibody conjugation. Methodsfor detecting such complexes, in addition to those described above forthe GST-immobilized complexes, include immunodetection of complexesusing antibodies reactive with the cancer marker protein or targetmolecule, as well as enzyme-linked assays which rely on detecting anenzymatic activity associated with the cancer marker protein or targetmolecule.

Alternatively, cell free assays can be conducted in a liquid phase. Insuch an assay, the reaction products are separated from unreactedcomponents, by any of a number of standard techniques, including, butnot limited to: differential centrifugation (see, for example, Rivas andMinton, Trends Biochem Sci 18:284-7 [1993]); chromatography (gelfiltration chromatography, ion-exchange chromatography); electrophoresis(see, e.g., Ausubel et al., eds. Current Protocols in Molecular Biology1999, J. Wiley: New York); and immunoprecipitation (see, for example,Ausubel et al., eds. Current Protocols in Molecular Biology 1999, J.Wiley: New York). Such resins and chromatographic techniques are knownto one skilled in the art (See e.g., Heegaard J. Mol. Recognit 11:141-8[1998]; Hage and Tweed J. Chromatogr. Biomed. Sci. Appl 699:499-525[1997]). Further, fluorescence energy transfer may also be convenientlyutilized, as described herein, to detect binding without furtherpurification of the complex from solution.

The assay can include contacting the cancer markers protein, mRNA, orbiologically active portion thereof with a known compound that binds thecancer marker to form an assay mixture, contacting the assay mixturewith a test compound, and determining the ability of the test compoundto interact with a cancer marker protein or mRNA, wherein determiningthe ability of the test compound to interact with a cancer markerprotein or mRNA includes determining the ability of the test compound topreferentially bind to cancer markers or biologically active portionthereof, or to modulate the activity of a target molecule, as comparedto the known compound.

To the extent that cancer markers can, in vivo, interact with one ormore cellular or extracellular macromolecules, such as proteins,inhibitors of such an interaction are useful. A homogeneous assay can beused can be used to identify inhibitors.

For example, a preformed complex of the target gene product and theinteractive cellular or extracellular binding partner product isprepared such that either the target gene products or their bindingpartners are labeled, but the signal generated by the label is quencheddue to complex formation (see, e.g., U.S. Pat. No. 4,109,496, hereinincorporated by reference, that utilizes this approach forimmunoassays). The addition of a test substance that competes with anddisplaces one of the species from the preformed complex will result inthe generation of a signal above background. In this way, testsubstances that disrupt target gene product-binding partner interactioncan be identified. Alternatively, cancer markers protein can be used asa “bait protein” in a two-hybrid assay or three-hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al., Cell 72:223-232 [1993]; Maduraet al., J. Biol. Chem. 268.12046-12054 [1993]; Bartel et al.,Biotechniques 14:920-924 [1993]; Iwabuchi et al., Oncogene 8:1693-1696[1993]; and Brent WO 94/10300; each of which is herein incorporated byreference), to identify other proteins, that bind to or interact withcancer markers (“cancer marker-binding proteins” or “cancer marker-bp”)and are involved in cancer marker activity. Such cancer marker-bps canbe activators or inhibitors of signals by the cancer marker proteins ortargets as, for example, downstream elements of a cancermarkers-mediated signaling pathway.

Modulators of cancer markers expression can also be identified. Forexample, a cell or cell free mixture is contacted with a candidatecompound and the expression of cancer marker mRNA or protein evaluatedrelative to the level of expression of cancer marker mRNA or protein inthe absence of the candidate compound. When expression of cancer markermRNA or protein is greater in the presence of the candidate compoundthan in its absence, the candidate compound is identified as astimulator of cancer marker mRNA or protein expression. Alternatively,when expression of cancer marker mRNA or protein is less (i.e.,statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of cancer marker mRNA or protein expression. The level ofcancer markers mRNA or protein expression can be determined by methodsdescribed herein for detecting cancer markers mRNA or protein.

A modulating agent can be identified using a cell-based or a cell freeassay, and the ability of the agent to modulate the activity of a cancermarkers protein can be confirmed in vivo, e.g., in an animal such as ananimal model for a disease (e.g., an animal with prostate cancer ormetastatic prostate cancer; or an animal harboring a xenograft of aprostate cancer from an animal (e.g., human) or cells from a cancerresulting from metastasis of a prostate cancer (e.g., to a lymph node,bone, or liver), or cells from a prostate cancer cell line.

This invention further pertains to novel agents identified by theabove-described screening assays (See e.g., below description of cancertherapies). Accordingly, it is within the scope of this invention tofurther use an agent identified as described herein (e.g., a cancermarker modulating agent, an antisense cancer marker nucleic acidmolecule, a siRNA molecule, a cancer marker specific antibody, or acancer marker-binding partner) in an appropriate animal model (such asthose described herein) to determine the efficacy, toxicity, sideeffects, or mechanism of action, of treatment with such an agent.Furthermore, novel agents identified by the above-described screeningassays can be, e.g., used for treatments as described herein.

VI. Therapeutic Applications

In some embodiments, the present invention provides therapies for cancer(e.g., prostate cancer). In some embodiments, therapies directly orindirectly target gene fusions of the present invention.

A. RNA Interference and Antisense Therapies

In some embodiments, the present invention targets the expression ofgene fusions. For example, in some embodiments, the present inventionemploys compositions comprising oligomeric antisense or RNAi compounds,particularly oligonucleotides (e.g., those identified in the drugscreening methods described above), for use in modulating the functionof nucleic acid molecules encoding cancer markers of the presentinvention, ultimately modulating the amount of cancer marker expressed.

1. RNA Interference (RNAi)

In some embodiments, RNAi is utilized to inhibit fusion proteinfunction. RNAi represents an evolutionary conserved cellular defense forcontrolling the expression of foreign genes in most eukaryotes,including humans. RNAi is typically triggered by double-stranded RNA(dsRNA) and causes sequence-specific mRNA degradation of single-strandedtarget RNAs homologous in response to dsRNA. The mediators of mRNAdegradation are small interfering RNA duplexes (siRNAs), which arenormally produced from long dsRNA by enzymatic cleavage in the cell.siRNAs are generally approximately twenty-one nucleotides in length(e.g. 21-23 nucleotides in length), and have a base-paired structurecharacterized by two nucleotide 3′-overhangs. Following the introductionof a small RNA, or RNAi, into the cell, it is believed the sequence isdelivered to an enzyme complex called RISC(RNA-induced silencingcomplex). RISC recognizes the target and cleaves it with anendonuclease. It is noted that if larger RNA sequences are delivered toa cell, RNase III enzyme (Dicer) converts longer dsRNA into 21-23 nt dssiRNA fragments. In some embodiments, RNAi oligonucleotides are designedto target the junction region of fusion proteins.

Chemically synthesized siRNAs have become powerful reagents forgenome-wide analysis of mammalian gene function in cultured somaticcells. Beyond their value for validation of gene function, siRNAs alsohold great potential as gene-specific therapeutic agents (Tuschl andBorkhardt, Molecular Intervent. 2002; 2 (3): 158-67, herein incorporatedby reference).

The transfection of siRNAs into animal cells results in the potent,long-lasting post-transcriptional silencing of specific genes (Caplen etal, Proc Natl Acad Sci U.S.A. 2001; 98: 9742-7; Elbashir et al., Nature.2001; 411:494-8; Elbashir et al., Genes Dev. 2001; 15: 188-200; andElbashir et al., EMBO J. 2001; 20: 6877-88, all of which are hereinincorporated by reference). Methods and compositions for performing RNAiwith siRNAs are described, for example, in U.S. Pat. No. 6,506,559,herein incorporated by reference.

siRNAs are extraordinarily effective at lowering the amounts of targetedRNA, and by extension proteins, frequently to undetectable levels. Thesilencing effect can last several months, and is extraordinarilyspecific, because one nucleotide mismatch between the target RNA and thecentral region of the siRNA is frequently sufficient to preventsilencing (Brummelkamp et al, Science 2002; 296:550-3; and Holen et al,Nucleic Acids Res. 2002; 30:1757-66, both of which are hereinincorporated by reference).

An important factor in the design of siRNAs is the presence ofaccessible sites for siRNA binding. Bahoia et al., (J. Biol. Chem.,2003; 278: 15991-15997; herein incorporated by reference) describe theuse of a type of DNA array called a scanning array to find accessiblesites in mRNAs for designing effective siRNAs. These arrays compriseoligonucleotides ranging in size from monomers to a certain maximum,usually Corners, synthesized using a physical barrier (mask) by stepwiseaddition of each base in the sequence. Thus the arrays represent a fulloligonucleotide complement of a region of the target gene. Hybridizationof the target mRNA to these arrays provides an exhaustive accessibilityprofile of this region of the target mRNA. Such data are useful in thedesign of antisense oligonucleotides (ranging from 7mers to 25mers),where it is important to achieve a compromise between oligonucleotidelength and binding affinity, to retain efficacy and target specificity(Sohail et al, Nucleic Acids Res., 2001; 29 (10): 2041-2045). Additionalmethods and concerns for selecting siRNAs are described for example, inWO 05054270, WO05038054A1, WO03070966A2, J Mol Biol. 2005 May 13; 348(4):883-93, J Mol Biol. 2005 May 13; 348 (4):871-81, and Nucleic AcidsRes. 2003 Aug. 1; 31 (15):4417-24, each of which is herein incorporatedby reference in its entirety. In addition, software (e.g., the MWGonline siMAX siRNA design tool) is commercially or publicly availablefor use in the selection of siRNAs.

2. Antisense

In other embodiments, fusion protein expression is modulated usingantisense compounds that specifically hybridize with one or more nucleicacids encoding cancer markers of the present invention. The specifichybridization of an oligomeric compound with its target nucleic acidinterferes with the normal function of the nucleic acid. This modulationof function of a target nucleic acid by compounds that specificallyhybridize to it is generally referred to as “antisense.” The functionsof DNA to be interfered with include replication and transcription. Thefunctions of RNA to be interfered with include all vital functions suchas, for example, translocation of the RNA to the site of proteintranslation, translation of protein from the RNA, splicing of the RNA toyield one or more mRNA species, and catalytic activity that may beengaged in or facilitated by the RNA. The overall effect of suchinterference with target nucleic acid function is modulation of theexpression of cancer markers of the present invention. In the context ofthe present invention, “modulation” means either an increase(stimulation) or a decrease (inhibition) in the expression of a gene.For example, expression may be inhibited to potentially prevent tumorproliferation.

It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of the present invention, is a multistep process. The processusually begins with the identification of a nucleic acid sequence whosefunction is to be modulated. This may be, for example, a cellular gene(or mRNA transcribed from the gene) whose expression is associated witha particular disorder or disease state, or a nucleic acid molecule froman infectious agent. In the present invention, the target is a nucleicacid molecule encoding a gene fusion of the present invention. Thetargeting process also includes determination of a site or sites withinthis gene for the antisense interaction to occur such that the desiredeffect, e.g., detection or modulation of expression of the protein, willresult. Within the context of the present invention, a preferredintragenic site is the region encompassing the translation initiation ortermination codon of the open reading frame (ORF) of the gene. Since thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). Eukaryotic and prokaryotic genes may have two or morealternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of the presentinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAmolecule transcribed from a gene encoding a tumor antigen of the presentinvention, regardless of the sequence(s) of such codons.

Translation termination codon (or “stop codon”) of a gene may have oneof three sequences (i.e., 5′-UAA, 5′-UAG and 5′-UGA; the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms“start codon region” and “translation initiation codon region” refer toa portion of such an mRNA or gene that encompasses from about 25 toabout 50 contiguous nucleotides in either direction (i.e., 5′ or 3′)from a translation initiation codon. Similarly, the terms “stop codonregion” and “translation termination codon region” refer to a portion ofsuch an mRNA or gene that encompasses from about 25 to about 50contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon.

The open reading frame (ORF) or “coding region,” which refers to theregion between the translation initiation codon and the translationtermination codon, is also a region that may be targeted effectively.Other target regions include the 5′ untranslated region (5′ UTR),referring to the portion of an mRNA in the 5′ direction from thetranslation initiation codon, and thus including nucleotides between the5′ cap site and the translation initiation codon of an mRNA orcorresponding nucleotides on the gene, and the 3′ untranslated region(3′ UTR), referring to the portion of an mRNA in the 3′ direction fromthe translation termination codon, and thus including nucleotidesbetween the translation termination codon and 3′ end of an mRNA orcorresponding nucleotides on the gene. The 5′ cap of an mRNA comprisesan N7-methylated guanosine residue joined to the 5′-most residue of themRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap. The cap region may also be apreferred target region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” that are excised from atranscript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. mRNA splice sites (i.e., intron-exonjunctions) may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred targets. It has also been found thatintrons can also be effective, and therefore preferred, target regionsfor antisense compounds targeted, for example, to DNA or pre-mRNA.

In some embodiments, target sites for antisense inhibition areidentified using commercially available software programs (e.g.,Biognostik, Gottingen, Germany; SysArris Software, Bangalore, India;Antisense Research Group, University of Liverpool, Liverpool, England;GeneTrove, Carlsbad, Calif.). In other embodiments, target sites forantisense inhibition are identified using the accessible site methoddescribed in PCT Publ. No. WO0198537A2, herein incorporated byreference.

Once one or more target sites have been identified, oligonucleotides arechosen that are sufficiently complementary to the target (i.e.,hybridize sufficiently well and with sufficient specificity) to give thedesired effect. For example, in preferred embodiments of the presentinvention, antisense oligonucleotides are targeted to or near the startcodon.

In the context of this invention, “hybridization,” with respect toantisense compositions and methods, means hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases. For example, adenine andthymine are complementary nucleobases that pair through the formation ofhydrogen bonds. It is understood that the sequence of an antisensecompound need not be 100% complementary to that of its target nucleicacid to be specifically hybridizable. An antisense compound isspecifically hybridizable when binding of the compound to the target DNAor RNA molecule interferes with the normal function of the target DNA orRNA to cause a loss of utility, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the antisense compoundto non-target sequences under conditions in which specific binding isdesired (i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and in the case of in vitro assays,under conditions in which the assays are performed).

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with specificity, can be used to elucidate thefunction of particular genes. Antisense compounds are also used, forexample, to distinguish between functions of various members of abiological pathway.

The specificity and sensitivity of antisense is also applied fortherapeutic uses. For example, antisense oligonucleotides have beenemployed as therapeutic moieties in the treatment of disease states inanimals and man. Antisense oligonucleotides have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides areuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues, and animals,especially humans.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric antisensecompounds, including but not limited to oligonucleotide mimetics such asare described below. The antisense compounds in accordance with thisinvention preferably comprise from about 8 to about 30 nucleobases(i.e., from about 8 to about 30 linked bases), although both longer andshorter sequences may find use with the present invention. Particularlypreferred antisense compounds are antisense oligonucleotides, even morepreferably those comprising from about 12 to about 25 nucleobases.

Specific examples of preferred antisense compounds useful with thepresent invention include oligonucleotides containing modified backbonesor non-natural internucleoside linkages. As defined in thisspecification, oligonucleotides having modified backbones include thosethat retain a phosphorus atom in the backbone and those that do not havea phosphorus atom in the backbone. For the purposes of thisspecification, modified oligonucleotides that do not have a phosphorusatom in their internucleoside backbone can also be considered to beoligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage (i.e., the backbone) of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science 254:1497 (1991).

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂, —NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂—, and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O- ,S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl andalkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. A preferred modificationincludes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta 78:486[1995]) i.e., an alkoxyalkoxy group. A further preferred modificationincludes 2′-dimethylaminooxyethoxy (i.e., a O(CH₂)₂ON(CH₃)₂ group), alsoknown as 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (also known in theart as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other preferred modifications include 2′-methoxy(2′-O—CH₃),2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808. Certainof these nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Another modification of the oligonucleotides of the present inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates that enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety, cholic acid,a thioether, (e.g., hexyl-S-tritylthiol), a thiocholesterol, analiphatic chain, (e.g., dodecandiol or undecyl residues), aphospholipid, (e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or apolyethylene glycol chain or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

One skilled in the relevant art knows well how to generateoligonucleotides containing the above-described modifications. Thepresent invention is not limited to the antisense oligonucleotidesdescribed above. Any suitable modification or substitution may beutilized.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds that are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of the presentinvention, are antisense compounds, particularly oligonucleotides, whichcontain two or more chemically distinct regions, each made up of atleast one monomer unit, i.e., a nucleotide in the case of anoligonucleotide compound. These oligonucleotides typically contain atleast one region wherein the oligonucleotide is modified so as to conferupon the oligonucleotide increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligonucleotide mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNaseH is a cellular endonuclease thatcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of oligonucleotide inhibition of geneexpression. Consequently, comparable results can often be obtained withshorter oligonucleotides when chimeric oligonucleotides are used,compared to phosphorothioate deoxyoligonucleotides hybridizing to thesame target region. Cleavage of the RNA target can be routinely detectedby gel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

Chimeric antisense compounds of the present invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above.

The present invention also includes pharmaceutical compositions andformulations that include the antisense compounds of the presentinvention as described below.

B. Gene Therapy

The present invention contemplates the use of any genetic manipulationfor use in modulating the expression of gene fusions of the presentinvention. Examples of genetic manipulation include, but are not limitedto, gene knockout (e.g., removing the fusion gene from the chromosomeusing, for example, recombination), expression of antisense constructswith or without inducible promoters, and the like. Delivery of nucleicacid construct to cells in vitro or in vivo may be conducted using anysuitable method. A suitable method is one that introduces the nucleicacid construct into the cell such that the desired event occurs (e.g.,expression of an antisense construct). Genetic therapy may also be usedto deliver siRNA or other interfering molecules that are expressed invivo (e.g., upon stimulation by an inducible promoter (e.g., anandrogen-responsive promoter)).

Introduction of molecules carrying genetic information into cells isachieved by any of various methods including, but not limited to,directed injection of naked DNA constructs, bombardment with goldparticles loaded with said constructs, and macromolecule mediated genetransfer using, for example, liposomes, biopolymers, and the like.Preferred methods use gene delivery vehicles derived from viruses,including, but not limited to, adenoviruses, retroviruses, vacciniaviruses, and adeno-associated viruses. Because of the higher efficiencyas compared to retroviruses, vectors derived from adenoviruses are thepreferred gene delivery vehicles for transferring nucleic acid moleculesinto host cells in vivo. Adenoviral vectors have been shown to providevery efficient in vivo gene transfer into a variety of solid tumors inanimal models and into human solid tumor xenografts in immune-deficientmice. Examples of adenoviral vectors and methods for gene transfer aredescribed in PCT publications WO 00/12738 and WO 00/09675 and U.S. Pat.Appl. Nos. 6,033,908, 6,019,978, 6,001,557, 5,994,132, 5,994,128,5,994,106, 5,981,225, 5,885,808, 5,872,154, 5,830,730, and 5,824,544,each of which is herein incorporated by reference in its entirety.

Vectors may be administered to subject in a variety of ways. Forexample, in some embodiments of the present invention, vectors areadministered into tumors or tissue associated with tumors using directinjection. In other embodiments, administration is via the blood orlymphatic circulation (See e.g., PCT publication 99/02685 hereinincorporated by reference in its entirety). Exemplary dose levels ofadenoviral vector are preferably 10⁸ to 10¹¹ vector particles added tothe perfusate.

C. Antibody Therapy

In some embodiments, the present invention provides antibodies thattarget prostate tumors that express a gene fusion of the presentinvention. Any suitable antibody (e.g., monoclonal, polyclonal, orsynthetic) may be utilized in the therapeutic methods disclosed herein.In preferred embodiments, the antibodies used for cancer therapy arehumanized antibodies. Methods for humanizing antibodies are well knownin the art (See e.g., U.S. Pat. Nos. 6,180,370, 5,585,089, 6,054,297,and 5,565,332; each of which is herein incorporated by reference).

In some embodiments, the therapeutic antibodies comprise an antibodygenerated against a gene fusion of the present invention, wherein theantibody is conjugated to a cytotoxic agent. In such embodiments, atumor specific therapeutic agent is generated that does not targetnormal cells, thus reducing many of the detrimental side effects oftraditional chemotherapy. For certain applications, it is envisionedthat the therapeutic agents will be pharmacologic agents that will serveas useful agents for attachment to antibodies, particularly cytotoxic orotherwise anticellular agents having the ability to kill or suppress thegrowth or cell division of endothelial cells. The present inventioncontemplates the use of any pharmacologic agent that can be conjugatedto an antibody, and delivered in active form. Exemplary anticellularagents include chemotherapeutic agents, radioisotopes, and cytotoxins.The therapeutic antibodies of the present invention may include avariety of cytotoxic moieties, including but not limited to, radioactiveisotopes (e.g., iodine-131, iodine-123, technicium-99m, indium-111,rhenium-188, rhenium-186, gallium-67, copper-67, yttrium-90, iodine-125or astatine-211), hormones such as a steroid, antimetabolites such ascytosines (e.g., arabinoside, fluorouracil, methotrexate or aminopterin;an anthracycline; mitomycin C), vinca alkaloids (e.g., demecolcine;etoposide; mithramycin), and antitumor alkylating agent such aschlorambucil or melphalan. Other embodiments may include agents such asa coagulant, a cytokine, growth factor, bacterial endotoxin or the lipidA moiety of bacterial endotoxin. For example, in some embodiments,therapeutic agents will include plant-, fungus- or bacteria-derivedtoxin, such as an A chain toxins, a ribosome inactivating protein,α-sarcin, aspergillin, restrictocin, a ribonuclease, diphtheria toxin orpseudomonas exotoxin, to mention just a few examples. In some preferredembodiments, deglycosylated ricin A chain is utilized.

In any event, it is proposed that agents such as these may, if desired,be successfully conjugated to an antibody, in a manner that will allowtheir targeting, internalization, release or presentation to bloodcomponents at the site of the targeted tumor cells as required usingknown conjugation technology (See, e.g., Ghose et al., Methods Enzymol.,93:280 [1983]).

For example, in some embodiments the present invention providesimmunotoxins targeted a cancer marker of the present invention (e.g.,ERG or ETV1 fusions). Immunotoxins are conjugates of a specifictargeting agent typically a tumor-directed antibody or fragment, with acytotoxic agent, such as a toxin moiety. The targeting agent directs thetoxin to, and thereby selectively kills, cells carrying the targetedantigen. In some embodiments, therapeutic antibodies employ crosslinkersthat provide high in vivo stability (Thorpe et al, Cancer Res., 48:6396[1988]).

In other embodiments, particularly those involving treatment of solidtumors, antibodies are designed to have a cytotoxic or otherwiseanticellular effect against the tumor vasculature, by suppressing thegrowth or cell division of the vascular endothelial cells. This attackis intended to lead to a tumor-localized vascular collapse, deprivingthe tumor cells, particularly those tumor cells distal of thevasculature, of oxygen and nutrients, ultimately leading to cell deathand tumor necrosis.

In preferred embodiments, antibody based therapeutics are formulated aspharmaceutical compositions as described below. In preferredembodiments, administration of an antibody composition of the presentinvention results in a measurable decrease in cancer (e.g., decrease orelimination of tumor).

D. Pharmaceutical Compositions

The present invention further provides pharmaceutical compositions(e.g., comprising pharmaceutical agents that modulate the expression oractivity of gene fusions of the present invention). The pharmaceuticalcompositions of the present invention may be administered in a number ofways depending upon whether local or systemic treatment is desired andupon the area to be treated. Administration may be topical (includingophthalmic and to mucous membranes including vaginal and rectaldelivery), pulmonary (e.g., by inhalation or insufflation of powders oraerosols, including by nebulizer; intratracheal, intranasal, epidermaland transdermal), oral or parenteral. Parenteral administration includesintravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; or intracranial, e.g., intrathecalor intraventricular, administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances that increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product.

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (WO 97/30731), also enhancethe cellular uptake of oligonucleotides.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antisense compounds and (b) one or more otherchemotherapeutic agents that function by a non-antisense mechanism.Examples of such chemotherapeutic agents include, but are not limitedto, anticancer drugs such as daunorubicin, dactinomycin, doxorubicin,bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatinand diethylstilbestrol (DES). Anti-inflammatory drugs, including but notlimited to nonsteroidal anti-inflammatory drugs and corticosteroids, andantiviral drugs, including but not limited to ribivirin, vidarabine,acyclovir and ganciclovir, may also be combined in compositions of theinvention. Other non-antisense chemotherapeutic agents are also withinthe scope of this invention. Two or more combined compounds may be usedtogether or sequentially.

Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient. Theadministering physician can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models or based on the examples described herein. Ingeneral, dosage is from 0.01 μg to 100 g per kg of body weight, and maybe given once or more daily, weekly, monthly or yearly. The treatingphysician can estimate repetition rates for dosing based on measuredresidence times and concentrations of the drug in bodily fluids ortissues. Following successful treatment, it may be desirable to have thesubject undergo maintenance therapy to prevent the recurrence of thedisease state, wherein the oligonucleotide is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight,once or more daily, to once every 20 years.

VII. Transgenic Animals

The present invention contemplates the generation of transgenic animalscomprising an exogenous cancer marker gene (e.g., gene fusion) of thepresent invention or mutants and variants thereof (e.g., truncations orsingle nucleotide polymorphisms). In preferred embodiments, thetransgenic animal displays an altered phenotype (e.g., increased ordecreased presence of markers) as compared to wild-type animals. Methodsfor analyzing the presence or absence of such phenotypes include but arenot limited to, those disclosed herein. In some preferred embodiments,the transgenic animals further display an increased or decreased growthof tumors or evidence of cancer.

The transgenic animals of the present invention find use in drug (e.g.,cancer therapy) screens. In some embodiments, test compounds (e.g., adrug that is suspected of being useful to treat cancer) and controlcompounds (e.g., a placebo) are administered to the transgenic animalsand the control animals and the effects evaluated.

The transgenic animals can be generated via a variety of methods. Insome embodiments, embryonal cells at various developmental stages areused to introduce transgenes for the production of transgenic animals.Different methods are used depending on the stage of development of theembryonal cell. The zygote is the best target for micro-injection. Inthe mouse, the male pronucleus reaches the size of approximately 20micrometers in diameter that allows reproducible injection of 1-2picoliters (pl) of DNA solution. The use of zygotes as a target for genetransfer has a major advantage in that in most cases the injected DNAwill be incorporated into the host genome before the first cleavage(Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442 [1985]). As aconsequence, all cells of the transgenic non-human animal will carry theincorporated transgene. This will in general also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene. U.S. Pat. No.4,873,191 describes a method for the micro-injection of zygotes; thedisclosure of this patent is incorporated herein in its entirety.

In other embodiments, retroviral infection is used to introducetransgenes into a non-human animal. In some embodiments, the retroviralvector is utilized to transfect oocytes by injecting the retroviralvector into the perivitelline space of the oocyte (U.S. Pat. No.6,080,912, incorporated herein by reference). In other embodiments, thedeveloping non-human embryo can be cultured in vitro to the blastocyststage. During this time, the blastomeres can be targets for retroviralinfection (Janenich, Proc. Natl. Acad. Sci. USA 73:1260 [1976]).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Hogan et al., in Manipulatingthe Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. [1986]). The viral vector system used to introduce thetransgene is typically a replication-defective retrovirus carrying thetransgene (Jahner et al., Proc. Natl. Acad. Sci. USA 82:6927 [1985]).Transfection is easily and efficiently obtained by culturing theblastomeres on a monolayer of virus-producing cells (Stewart, et al.,EMBO J., 6:383 [1987]). Alternatively, infection can be performed at alater stage. Virus or virus-producing cells can be injected into theblastocoele (Jahner et al., Nature 298:623 [1982]). Most of the founderswill be mosaic for the transgene since incorporation occurs only in asubset of cells that form the transgenic animal. Further, the foundermay contain various retroviral insertions of the transgene at differentpositions in the genome that generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into thegermline, albeit with low efficiency, by intrauterine retroviralinfection of the midgestation embryo (Jahner et al., supra [1982]).Additional means of using retroviruses or retroviral vectors to createtransgenic animals known to the art involve the micro-injection ofretroviral particles or mitomycin C-treated cells producing retrovirusinto the perivitelline space of fertilized eggs or early embryos (PCTInternational Application WO 90/08832 [1990], and Haskell and Bowen,Mol. Reprod. Dev., 40:386 [1995]).

In other embodiments, the transgene is introduced into embryonic stemcells and the transfected stem cells are utilized to form an embryo. EScells are obtained by culturing pre-implantation embryos in vitro underappropriate conditions (Evans et al., Nature 292:154 [1981]; Bradley etal, Nature 309:255 [1984]; Gossler et al., Proc. Acad. Sci. USA 83:9065[1986]; and Robertson et al., Nature 322:445 [1986]). Transgenes can beefficiently introduced into the ES cells by DNA transfection by avariety of methods known to the art including calcium phosphateco-precipitation, protoplast or spheroplast fusion, lipofection andDEAE-dextran-mediated transfection. Transgenes may also be introducedinto ES cells by retrovirus-mediated transduction or by micro-injection.Such transfected ES cells can thereafter colonize an embryo followingtheir introduction into the blastocoel of a blastocyst-stage embryo andcontribute to the germ line of the resulting chimeric animal (forreview, See, Jaenisch, Science 240:1468 [1988]). Prior to theintroduction of transfected ES cells into the blastocoel, thetransfected ES cells may be subjected to various selection protocols toenrich for ES cells which have integrated the transgene assuming thatthe transgene provides a means for such selection. Alternatively, thepolymerase chain reaction may be used to screen for ES cells that haveintegrated the transgene. This technique obviates the need for growth ofthe transfected ES cells under appropriate selective conditions prior totransfer into the blastocoel.

In still other embodiments, homologous recombination is utilized toknock-out gene function or create deletion mutants (e.g., truncationmutants). Methods for homologous recombination are described in U.S.Pat. No. 5,614,396, incorporated herein by reference.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1 ERG and ETV1 Gene Fusions A. Materials and Methods CancerOutlier Profile Analysis (COPA)

COPA analysis was performed on 132 gene expression data sets in Oncomine3.0 comprising 10,486 microarray experiments. In addition, data from 99amplified laser-capture microdissected prostate tissue samples wereincluded in the COPA analysis. COPA has three steps. First, geneexpression values are median centered, setting each gene's medianexpression value to zero. Second, the median absolute deviation (MAD) iscalculated and scaled to 1 by dividing each gene expression value by itsMAD. Median and MAD were used for transformation as opposed to mean andstandard deviation so that outlier expression values do not undulyinfluence the distribution estimates, and are thus preservedpost-normalization. Third, the 75th, 90th, and 95th percentiles of thetransformed expression values are tabulated for each gene and then genesare rank-ordered by their percentile scores, providing a prioritizedlist of outlier profiles.

Samples

Tissues utilized were from the radical prostatectomy series at theUniversity of Michigan and from the Rapid Autopsy Program (Shah et al.,Cancer Res 64, 9209 (Dec. 15, 2004)), which are both part of Universityof Michigan Prostate Cancer Specialized Program of Research Excellence(S.P.O.R.E.) Tissue Core.

Tissues were also obtained from a radical prostatectomy series at theUniversity Hospital Ulm (Ulm, Germany). All samples were collected fromconsented patients with prior institutional review board approval ateach respective institution. Total RNA from all samples was isolatedwith Trizol (Invitrogen) according to the manufacturer's instructions.Total RNA was also isolated from RWPE, PC3, PC3+AR (Dai et al., Steroids61, 531 (1996)), LNCaP, VCaP and DuCaP cell lines. RNA integrity wasverified by denaturing formaldehyde gel electrophoresis or the AgilentBioanalyzer 2100. A commercially available pool of benign prostatetissue total RNA (CPP, Clontech) was also used.

Quantitative PCR (QPCR)

Quantitative PCR (QPCR) was performed using SYBR Green dye on an AppliedBiosystems 7300 Real Time PCR system essentially as described(Chinnaiyan et al., Cancer Res 65, 3328 (2005); Rubin et al., Cancer Res64, 3814 (2004)). Briefly, 1-5 μg of total RNA was reverse transcribedinto cDNA using SuperScript III (Invitrogen) in the presence of randomprimers or random primers and oligo dT primers. All reactions wereperformed with SYBR Green Master Mix (Applied Biosystems) and 25 ng ofboth the forward and reverse primer using the manufacturer's recommendedthermocycling conditions. All reactions were subjected to melt curveanalysis and products from selected experiments were resolved byelectrophoreses on 1.5% agarose gels. For each experiment, thresholdlevels were set during the exponential phase of the QPCR reaction usingSequence Detection Software version 1.2.2 (Applied Biosystems). Theamount of each target gene relative to the housekeeping geneglyceraldehyde-3-phosphate dehydrogenase (GAPDH) for each sample wasdetermined using the comparative threshold cycle (Ct) method (AppliedBiosystems User Bulletin #2), with the cDNA sample serving as thecalibrator for each experiment described in the figure legend. Alloligonucleotide primers were synthesized by Integrated DNA Technologies.GAPDH primers were as described (Vandesompele et al., Genome Biol 3,RESEARCH0034 (2002)) and all other primers are listed (Table 4).

Approximately equal efficiencies of the primers were confirmed usingserial dilutions of prostate cancer cDNA or plasmid templates in orderto use the comparative Ct method.

RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE)

RNA ligase mediated rapid amplification of cDNA ends was performed usingthe GeneRacer RLM-RACE kit (Invitrogen), according to the manufacturer'sinstructions. Initially, samples were selected based on expression ofERG or ETV1 by QPCR. Five micrograms of total RNA was treated with calfintestinal phosphatase to remove 5′ phosphates from truncated mRNA andnon-mRNA and decapped with tobacco acid phyrophosphatase. The GeneRaceRNA Oligo was ligated to full length transcripts and reverse transcribedusing SuperScript III. To obtain 5′ ends, first-strand cDNA wasamplified with Platinum Taq High Fidelity (Invitrogen) using theGeneRacer 5′ Primer and ETV1 exon 4-5_r for ETV1 or the GeneRacer 5′Primer and ERG exon 4a_r or ERG exon 4b_r for ERG. Primer sequences aregiven (Table S2). Products were resolved by electrophoresis on 1.5%agarose gels and bands were excised, purified and TOPO TA cloned intopCR 4-TOPO. Purified plasmid DNA from at least 4 colonies was sequencedbi-directionally using M13 Reverse and M13 Forward (−20) primers or T3and T7 primers on an ABI Model 3730 automated sequencer by theUniversity of Michigan DNA Sequencing Core. RLM-RACEd cDNA was not usedfor the other assays.

Reverse-Transcription PCR for TMPRSS2:ERG Fusion

After identifying TMPRSS2:ERG positive cases using QPCR as describedabove, the same cDNA samples were PCR amplified with Platinum Taq HighFidelity and TPRSS2:ERG primers. Products were resolved byelectrophoresis, cloned into pCR 4-TOPO and sequenced as describedabove.

In Vitro Androgen Responsiveness

RWPE, LNCaP, VCap DuCaP, PC3 and PC3 cells stably transfected with thehuman androgen receptor (PC3+AR) (3) were treated for 24 h with 1%ethanol control or 1 nM of the synthetic androgen R1881. Total RNA wasisolated and subjected to reverse transcription and QPCR as describedabove with ERG exon 5-6 f and r primers. The relative amount ofERG/GAPDH for each sample was calibrated to the RWPE control sample.

Fluorescence In Situ Hybridization (FISH)

Formalin-fixed paraffin-embedded (FFPE) tissue sections from normalperipheral lymphocytes and the metastatic prostate cancer samples MET-26and MET-28 were used for interphase fluorescence in situ hybridization(FISH) analysis. In addition, interphase FISH was performed on a tissuemicroarray containing cores from FFPE sections of 13 clinicallylocalized prostate cancer and 16 metastatic prostate cancer samples. Atwo-color, two-signal approach was employed to evaluate the fusion ofTMPRSS2 and ETV1, with probes spanning most of the respective gene loci.The biotin-14-dCTP BAC clone RP11-124L22 was used for the ETV1 locus andthe digoxin-dUTP lableled BAC clone RPP11-35CD was used for the TMPRSS2locus. For analyzing gene rearrangements involving ERG, a split-signalprobe strategy was used, with two probes spanning the ERG locus(biotin-14-dCTP labeled BAC clone RP11-476D17 and digoxin-dUTP labeledBAC clone RP11-95I21). All BAC clones were obtained from the Children'sHospital of Oakland Research Institute (CHORI). Prior to tissueanalysis, the integrity and purity of all probes were verified byhybridization to metaphase spreads of normal peripheral lymphocytes.Tissue hybridization, washing and color detection were performed asdescribed (Rubin et al., Cancer Res 64, 3814 (2004); Garraway et al.,Nature 436, 117 (2005)).

B. Results Cancer Outlier Profile Analysis

In recent years, gene expression profiling with DNA microarrays hasbecome a common method to study the cancer transcriptome. Microarraystudies have provided great insight into the molecular heterogeneity ofcancer, often identifying novel molecular subtypes of disease thatcorrespond to tumor histology, patient outcome, and treatment response(Valk et al., N Engl J Med 350, 1617 (2004)). However, in general,transcriptome analysis has not led to the discovery of novel causalcancer genes. It was hypothesized that rearrangements and high levelcopy number changes that result in marked over-expression of an oncogeneshould be evident in transcriptome data, but not necessarily bytraditional analytical approaches.

In the majority of cancer types, heterogeneous patterns of oncogeneactivation have been observed, thus traditional analytical methods thatsearch for common activation of genes across a class of cancer samples(e.g., t-test or signal-to-noise ratio) will fail to find such oncogeneexpression profiles. Instead, a method that searches for markedover-expression in a subset of cases is needed. Experiments conductedduring the course of development of the present invention resulted inthe development of Cancer Outlier Profile Analysis (COPA). COPA seeks toaccentuate and identify outlier profiles by applying a simple numericaltransformation based on the median and median absolute deviation of agene expression profile (Ross et al., Blood 102, 2951 (2003)). Thisapproach is illustrated in FIG. 5A. COPA was applied to the Oncominedatabase (Bittner et al., Nature 406, 536 (2000)), which comprised acompendium of 132 gene expression datasets representing 10,486microarray experiments. COPA correctly identified several outlierprofiles for genes in specific cancer types in which a recurrentrearrangement or high-level amplification is known to occur. Theanalysis was focused on outlier profiles of known causal cancer genes,as defined by the Cancer Gene Census (Vasselli et al., Proc Natl AcadSci USA 100, 6958 (2003)), that ranked in the top 10 outlier profiles inan Oncomine dataset (Table 1 and Table 3). For example, in the Valk etal. acute myeloid leukemia (AML) dataset, RUNX1T1 (ETO) had thestrongest outlier profile at the 95th percentile, consistent with thisgene's known translocation and oncogenic activity in a subset of AML(Davis et al., Proc Natl Acad Sci USA 100, 6051 (2003)) (Table 1). Theoutlier profile precisely associated with cases that had a documentedt(8;21) translocation which fuses RUNX1 (AML1) and RUNX1T1 (ETO) (FIG.5B). Similarly, in the Ross et al. acute lymphoblastic leukemia (ALL)dataset, PBX1 showed the strongest outlier profile at the 90thpercentile, consistent with the E2A-PBX1 translocation known to occur ina subset of ALL (Segal et al., J Clin Oncol 21, 1775 (2003)) (Table 1).Again, the outlier expression profile perfectly correlated with thecharacterized t(1;19) E2A-PBX1 translocation in this panel of ALLs (FIG.S1C).

Identification of Outlier Profiles for ETS Family Members ERG and ETV1in Prostate Cancer

Novel COPA predictions were next examined. In several independentdatasets, COPA identified strong outlier profiles in prostate cancer forERG and ETV1, two ETS family transcription factors that are known to beinvolved in oncogenic translocations in Ewing's sarcoma and myeloidleukemias (Lapointe et al., Proc Natl Acad Sci USA 101, 811 (2004); Tianet al., N Engl J Med 349, 2483 (2003)). In the Dhanasekaran et al.(Keats et al., Blood 105, 4060 (2005)), Welsh et al. (Dhanasekaran etal., Faseb J 19, 243 (2005)) and Lapointe et al. (Wang et al., Lancet365, 671 (2005)) prostate cancer gene expression datasets, ERG had thehighest scoring outlier profile at the 75th percentile (Table 1), whilein the Lapointe et al. and Tomlins et al. (Welsh et al., Cancer Res 61,5974 (2001)) datasets, ETV1 had the highest scoring outlier profile atthe 90th percentile (Table 1). In total, COPA ranked ERG or ETV1 withinthe top ten outlier genes nine times in seven independent prostatecancer profiling studies. Both ERG and ETV1 are involved in oncogenictranslocations in Ewing's sarcoma. Fusion of the 5′ activation domain ofthe EWS gene to the highly conserved 3′ DNA binding domain of an ETSfamily member, such as ERG (t(21;22)(q22;q12)) or ETV1(t(7;22)(p21;q12)), is characteristic of Ewing's sarcoma (Lapoint etal., supra; Zhan et al., Blood 99, 1745 (2002); Fonseca et al., CancerRes 64, 1546 (2004)). Because translocations involving ETS familymembers are functionally redundant in oncogenic transformation, only onetype of translocation is typically observed in each case of Ewing'ssarcoma.

It was contemplated that if ERG and ETV1 are similarly involved in thedevelopment of prostate cancer, their outlier profiles should bemutually exclusive, that is, each case should over-express only one ofthe two genes. Mutations in functionally redundant genes, or genes inthe same oncogenic pathway, are unlikely to be co-selected for inneoplastic progression. The joint expression profiles of ERG and ETV1was examined across several prostate cancer datasets and it was foundthat they showed mutually exclusive outlier profiles. ERG and ETV1expression profiles from two large-scale transcriptome studies (Wang etal., supra; Cheok et al., Nat Genet 34, 85 (2003)), which profiledgrossly dissected prostate tissues using different microarray platforms,were identified (FIG. 1A, left and middle panels). The study by Lapointeet al. profiled benign prostate tissue, clinically localized prostatecancer, and metastatic prostate cancer, with ERG and ETV1 outlierexpression restricted to prostate cancer and metastatic prostate cancer,while the study by Glinsky et al. profiled clinically localized prostatecancer samples only. In both studies, prostate cancers exclusivelyexpressed ERG or ETV1 (FIG. 1A, right panel). Similar results were foundin a profiling study of 99 prostate tissue samples obtained by lasercapture microdissection (LCM) (Welsh et al., supra). In addition toexclusive outlier expression of either ERG or ETV1 (FIG. 1B, rightpanel), results from the LCM study demonstrated that ETV1 and ERG areonly over-expressed in epithelial cells from prostate cancer ormetastatic prostate cancer, but not in the putative precursor lesionprostatic intraepithelial neoplasia (PIN) or adjacent benign epithelia.To directly determine whether the observed exclusive outlier pattern isconsistent with other translocations where an activating gene can fusewith multiple partners, the Zhan et al. multiple myeloma dataset(Dhanasekaran et al., Nature 412, 822 (2001)) was examined. Recurrentfusions of the immunoglobulin heavy chain promoter to CCND1 or FGFR3,t(11,14) or t(4,14) respectively, characterize specific subsets ofmultiple myeloma (Wigle et al., Cancer Res 62, 3005 (2002)). Thesetranslocations were reflected in the outlier profile analysis (FIG. 1C),as CCND1 was the highest scoring outlier at the 75th percentile andFGFR3 was the third highest scoring outlier at the 95th percentile(Table 1). Except for two cases, myeloma samples showed exclusiveover-expression of CCND1 or FGFR3 (FIG. 1C, right panel). Takentogether, the outlier profiles of ERG and ETV1 across multiple prostatecancer data sets are consistent with other causal mutations in varioushuman malignancies. The exclusive over-expression of ERG or ETV1 inindividual prostate cancer samples is consistent with other neoplasms inwhich an activating gene can fuse with biologically redundant partnergenes, such as in multiple myeloma.

Discovery of a Recurrent Gene Fusion of TMPRSS2 to ERG or ETV1 inProstate Cancer.

The mechanism of ERG and ETV1 over-expression in individual prostatecancer samples was next determined. Prostate cancer cell lines andclinical specimens that over-expressed ERG or ETV1 were identified byperforming quantitative PCR (QPCR) (FIG. 2A). The LNCaP prostate cancercell line and two specimens obtained from a patient who died of hormonerefractory metastatic prostate cancer (MET-26RP, residual primarycarcinoma in the prostate and MET-26LN, a lymph node metastasis)markedly over-expressed ETV1 by QPCR (FIG. 2A). Five independentmetastatic foci from different anatomical locations as well as theresidual carcinoma in the prostate from this patient also over-expressedETV1 by DNA microarray analysis (Welsh et al., supra), suggesting thatETV1 activation occurred in the primary tumor before widespreadmetastasis. A lymph node metastasis was also identified from a secondpatient who died of hormone refractory metastatic prostate cancer(MET-28LN) and two prostate cancer cell lines, VCaP and DuCaP, thatover-expressed ERG (FIG. 2A). These cell lines were independentlyisolated from a vertebral metastasis (VCaP) and a dural metastasis(DuCaP) from a third patient with hormone-refractory prostate cancer(Golub et al., Science 286, 531 (1999); Rosenwald et al., Cancer Cell 3,185 (2003)). The common over-expression of ERG in these two cell linesagain suggests that ERG activation occurred before widespreadmetastasis. Taken together, these results suggest that specific geneticevents may activate ERG or ETV1 in individual samples during prostatetumorigenesis.

In an effort to characterize these genetic events, samples with high ERGor ETV1 expression were tested for chromosomal amplifications at theirrespective loci (7p21.2 and 21q22.3). By QPCR on genomic DNA,amplification of ERG or ETV1 in samples with respective transcriptover-expression (Sotiriou et al., Proc Natl Acad Sci USA 100, 10393(2003)) was not found. Next, the occurrence of DNA rearrangements wasassayed. Because the primers used for the QPCR described above werelocated 5′ to the known breakpoints for ERG and ETV1 in Ewing's sarcoma,it was unlikely that the same translocations occur in prostate cancer.Accordingly, the expression level of ETV1 exons was measured by exonwalking QPCR in the samples identified above that displayed ETV1over-expression. Five primer pairs spanning ETV1 exons 2 through 7 wereused and LNCaP cells showed essentially uniform over-expression of allmeasured ETV1 exons, and both MET26 specimens showed >90% reduction inthe expression of ETV1 exons 2 and 3 compared to exons 4-7 (FIG. 2B).Potential explanations for this result include alternative splicing, anovel cancer-specific isoform or an unreported rearrangement.

In order to characterize the full length ETV1 transcript, 5′ RNAligase-mediated rapid amplification of cDNA ends (RLM-RACE) wasperformed on LNCaP cells and MET26-LN. In addition, RLM-RACE wasperformed to obtain the full length transcript of ERG in MET28-LN. ForPCR amplification of ETV1 from the RLM-RACE cDNA, a forward primercomplementary to the RNA-oligonucleotide ligated to the 5′ end ofcomplete transcripts and a reverse primer in exon 4, the 5′-most exonthat was over-expressed in both LNCaP cells and MET26-LN was used.Utilizing a similar strategy as described above, it was determined thatexon 4 of ERG was over-expressed in MET28-LN. A reverse primer in thisexon was utilized for PCR amplification of RLM-RACE cDNA. Sequencing ofthe cloned products revealed fusions of the prostate specific geneTMPRSS2 (28) (21q22.2) with ETV1 in MET26-LN and with ERG in MET28-LN(FIG. 2C). In MET26-LN, two RLM-RACE PCR products were identified.

The first product, TMPRSS2:ETV1a, resulted in a fusion of the completeexon 1 of TMPRSS2 with the beginning of exon 4 of ETV1 (FIG. 2C). Thesecond product, TMPRSS2:ETV1b, resulted in a fusion of exons 1 and 2 ofTMPRSS2 with the beginning of exon 4 of ETV1 (FIG. 6). Both products areconsistent with the exon-walking QPCR described above, where MET26-LNshowed loss of over-expression in exons 2 and 3. In MET28-LN, a singleRLM-RACE PCR product was identified and sequencing revealed a fusion ofthe complete exon 1 of TMPRSS2 with the beginning of exon 4 of ERG(TMPRSS2:ERGa) (FIG. 2C).

Validation of TMPRSS2:ERG and TMPRSS2:ETV1 Gene Fusions in ProstateCancer

Based on these results, QPCR primer pairs were designed with forwardprimers in TMPRSS2 and reverse primers in exon 4 of ERG and ETV1. SYBRGreen QPCR was performed using both primer pairs across a panel ofsamples from 42 cases of clinically localized prostate cancer andmetastatic prostate cancer, with representative results depicted (FIGS.2, D and E). These results demonstrate that only samples with highlevels of ETV1 or ERG express the respective fusion product withTMPRSS2. Although QPCR resulted in measurable product after 35 cycles insome negative samples, melt curve analysis revealed distinct products inpositive and negative samples, and gel electrophoresis of products afterthe 40 cycle QPCR analysis revealed only primer dimmers in negativefusion samples (FIGS. 2, D and E). The formation of primer dimers may inpart be explained by the difficulty in designing primers entirely inexon 1 of TMPRSS2 due to the high GC content (80.3%). However, thespecific expression of TMPRSS2:ERGa, TMPRSS2:ETV1a and TMPRSS2:ETV1bfusions was confirmed using Taqman QPCR, with the forward primerspanning the respective fusion, and in each case, products were onlydetected in the same cases as the SYBR Green QPCR (Sotiriou et al.,supra). To further confirm the specificity of the primers used for SYBRGreen QPCR and the amplicons, standard reverse-transcription PCR wasperformed with the same primers as the SYBR Green QPCR on a panel ofsamples that expressed TMPRSS2:ERGa. Similar sized products wereobtained and sequencing of cloned products confirmed the presence ofTMPRSS2:ERGa. Two cases, PCA16 and PCA17, which expressed high levels ofETV1 or ERG respectively, but showed no evidence of the translocation byQPCR (FIGS. 2, D and E) were identified. RLM-RACE supported theseresults, as sequencing of the product produced with ETV1 primers inPCA16 revealed no evidence of a fusion transcript and no product couldbe obtained with ERG primers in PCA17. Similar results were obtained forLNCaP cells, with no evidence of a fusion by RLMRACE or QPCR, consistentwith the exon walking QPCR described above.

Summary of Evidence for TMPRSS2 Fusion Transcripts with ETS FamilyMembers in Prostate Cancer Samples

Results from three different assays for the TMPRSS2:ERG and TMPRSS2:ETV1fusion transcripts including sequencing of RLM-RACE products, QPCR andsequencing of RT-PCR products are summarized in Table 2. In addition toQPCR for TMPRSS2 fusions being performed in all samples, the existenceof these fusions was confirmed using several techniques on selectedsamples. For example, in PCA1 (prostate cancer sample 1), TMPRSS2:ERGawas identified using sequencing of RLMRACE products, QPCR and sequencingof RT-PCR products. By QPCR melt curve analysis and gel electrophoresisof QPCR products, PCA4 produced a larger amplicon than expected.Subsequent RLM-RACE analysis confirmed a fusion of the complete exon 1of TMPRSS2 with the beginning of exon 2 of ERG (TMPRSS2:ERGb) (FIG. 6).Taqman QPCR with the forward primer spanning the TMPRSS2:ERGb junctionconfirmed the presence of TMPRSS2:ERGb only in PCA4 and Taqman QPCR withthe forward primer spanning the TMPRSS2:ERGa junction did not produce aproduct in this specimen (27). Evidence for the TMPRSS2:ERG andTMPRSS2:ETV1 fusions were only found in cases that over-expressed ERG orETV1 respectively, by QPCR or DNA microarray. These results are inagreement with the exclusive expression observed in the outlieranalysis.

Fluorescence In Situ Hybridization (FISH) Confirms TMPRSS2:ETV1Translocation and ERG Rearrangement

After confirming the existence of the TMPRSS2:ETV1 and TMPRSS2:ERGfusion transcripts, evidence of these rearrangements at the chromosomallevel was obtained using interphase fluorescence in situ hybridization(FISH) on formalin fixed paraffin embedded (FFPE) specimens. Twodifferent probe strategies were employed: a two color, fusion-signalapproach to detect TMPRSS2:ETV1 translocations and a two-color,split-signal approach to detect rearrangements of the ERG locus. Theseprobe strategies were validated on the two cases initially used forRLM-RACE, MET26 and MET28 (FIG. 3). Using probes for TMPRSS2 and ETV1,normal peripheral lymphocytes (NPLs) demonstrated a pair of red and apair of green signals (FIG. 3A). MET26 showed fusion of one pair ofsignals, indicative of probe overlap (FIG. 3B, yellow arrowhead),consistent with the expression of the TMPRSS2:ETV1 transcript in thissample. In addition, consistent low-level amplification of the ETV1locus was identified, as indicated by the two remaining signals for ETV1(FIG. 3B, red arrowheads). Similarly, using probes spanning the 5′ and3′ region of the ERG locus, a pair of yellow signals in NPLs wasobserved (FIG. 3C). In MET28, one pair of probes split into separategreen and red signals, indicative of a rearrangement at the ERG locus(FIG. 3D, green and red arrows). This result is consistent with theexpression of the TMPRSS2:ERG transcript in this case. Based on theseresults, the individual FISH analyses described above were performed onserial tissue microarrays containing cores from 13 cases of localizedprostate cancer and 16 cases of metastatic prostate cancer (FIG. 3E). Asindicated by the matrix, 23 of 29 cases (79.3%) showed evidence ofTMPRSS2:ETV1 fusion (7 cases) or ERG rearrangement (16 cases). Inaddition, 12 of 29 cases (41.4%) showed evidence of low levelamplification at the ETV1 locus. Previous reports have identified thegenomic location of ETV1, 7p, as one of the most commonly amplifiedregions in localized and metastatic prostate cancer (Slamon et al.,Science 235, 177 (1987)). However it does not appear that 7pamplification drives ETV1 expression, as ETV1 amplification occurred in6 cases with ERG rearrangements and our transcript data demonstratesthat 0 of 19 samples with high ERG expression and the TMPRSS2:ERG fusionalso have high ETV1 expression. Furthermore, when both ETV1amplification and the TMPRSS2:ETV1 fusion were present by FISH, only theindividual ETV1 signal was amplified and not the fused signal.Nevertheless, results from this FISH analysis demonstrate the presenceof TMPRSS2:ETV1 and ERG rearrangements at the genomic level consistentwith the transcript data described above.

TMPRSS2 is an androgen-regulated gene and fusion with ERG results inandrogen regulation of ERG. TMPRSS2 was initially identified as aprostate-specific gene whose expression was increased by androgen inLNCaP cells and also contains androgen responsive elements (AREs) in itspromoter (Huang et al., Lancet 361, 1590 (2003); Schwartz et al., CancerRes 62, 4722 (2002)). Subsequent studies have confirmed high expressionin normal and neoplastic prostate tissue and demonstrated that TMPRSS2is androgen-regulated in androgen-sensitive prostate cell lines(Schwartz et al., Cancer Res 62, 4722 (2002); Ferrando et al., CancerCell 1, 75 (2002); Chen et al., Mol Biol Cell 14, 3208 (2003); LaTulippeet al., Cancer Res 62, 4499 (2002)). In addition, while androgen doesnot increase the expression of TMPRSS2 in the androgen insensitiveprostate cancer cell line PC3, stable expression of the androgenreceptor in PC3 cells resulted in TMPRSS2 becoming androgen responsive(Schwartz et al., supra; Ferrando et al., supra; Chen et al., supra;LaTulippe et al., supra). In contrast, microarray studies of LNCaPprostate cell lines treated with androgen have not identified ERG orETV1 as being androgen-responsive (Jain et al., Cancer Res 64, 3907(2004)) and examination of their promoter sequences did not revealconsensus AREs (Sotiriou et al., supra). It was contemplated that theTMPRSS2:ERGa fusion in DuCaP and VCaP cell lines, which was confirmed bythree independent assays in each cell line (Table 2), would result inthe androgen regulation of ERG. Using QPCR to assay for ERG expression,it was confirmed that even though ERG was highly expressed in both VCaPand DuCaP cells, treatment with the synthetic androgen R1881 increasedthe expression of ERG 2.57 fold in DuCaP cells and 5.02 fold in VCaPcells compared to untreated controls (FIG. 4). Expression of ERG wasminimal and essentially unchanged after R1881 treatment in RWPE (1.37fold), LnCaP (0.86 fold), PC3 (1.28 fold) and PC3 cells expressing theandrogen receptor (0.73 fold) compared to untreated controls.

Microarray analysis of the same samples confirmed that ERG was onlyup-regulated in response to androgen in DuCaP and VCaP cells (Sotiriouet al., supra). The present invention is not limited to a particularmechanism. Indeed, an understanding of the mechanism is not necessary topractice the present invention. Nonetheless, it is contemplated thatthese results suggest a possible mechanism for the aberrant expressionof ERG or ETV1 in prostate cancer when respective fusions with TMPRSS2are present.

TABLE 1 Table 1. Cancer Outlier Profile Analysis (COPA). Genes known toundergo causal mutations in cancer that had strong outlier profiles.Rank % Score Study Cancer Gene Evidence 1 95 20.056 Valk et al., N EnglJ Leukemia RUNX1T1 XX Med 350, 1617 (2004) 1 95 15.4462 Vasselli et al.,Renal PRO1073 X PNAS USA 100, 6958 (2003) 1 90 12.9581 Ross et al.,Blood Leukemia PBX1 XX 102, 2951 (2003). 1 95 10.03795 Lapointe et al.,Prostate ETV1 ** PNAS USA 101, 811 (Jan. 20, 2004) 1 90 9.1163 ProstateETV1 ** 1 90 7.4557 Tian et al., N Engl J Myeloma WHSC1 X Med 349, 2483(2003) 1 75 5.4071 Dhanasekaran et al., Prostate ERG ** Nature 412, 822(2001) 1 75 4.3628 Welsh et al., Cancer Prostate ERG ** Res 61, 5974(2001) 1 75 4.3425 Zhan et al., Blood Myeloma CCND1 X 99, 1745 (2002) 175 3.4414 Lapointe et al., Prostate ERG ** supra 1 75 3.3875Dhanasekaran et al., Prostate ERG ** Faseb J 19, 243 (2005) 2 90 6.7029Prostate ERG ** 3 95 13.3478 Zhan et al., supra Myeloma FGFR3 X 4 752.5728 Huang et al., Lancet Breast ERBB2 Y 361, 1590 (2003) 6 90 6.6079Sotiriou et al., Breast ERBB2 Y PNAS USA 100, 10393 (2003) 9 95 17.1698Glinsky et al., J Prostate ETV1 ** Clin Invest 113, 913 (2004) 9 906.60865 Nielsen et al., Sarcoma SSX1 X Lancet 359, 1301 (2002) 9 752.2218 Yu et al., J Clin Prostate ERG ** Oncol 22, 2790 (2004) “X”,signifies literature evidence for acquired pathogenomic translocation.“XX” signifies literature evidence for the specific translocation aswell as the samples in the specific study that were characterized forthat translocation. “Y” signifies consistent with known amplification.“**” signifies ERG and ETV1 outlier profiles in prostate cancer.Table 2 shows a summary of TMPRSS2 fusion to ETS family member status inprostate cancer samples and cell lines. For all assays, positive resultsare indicated by “+” and negative results are indicated by “−”. Blankcells indicate that the specific assay was not performed for thatsample.

Over-Expression of ERG or ETV1 by Quantitative PCR

(QPCR) is indicated and samples marked with an asterisk indicate thesample was also assessed by cDNA microarray and over-expression wasconfirmed. In order to detect TMPRSS2:ERG or TMPRSS2:ETV1 gene fusions,selected samples were subjected to RLM-RACE for the over-expressed ETSfamily member and samples with the TMPRSS2 fusion after sequencing areindicated. All samples were assayed for TMPRSS2:ETV1 and TMPRSS2:ERGexpression by QPCR. Selected cases were also amplified by standardreverse-transcription PCR (RT-PCR) using the same TMPRSS2 fusion primersas for QPCR and amplicons were sequenced. Samples with evidence forTMPRSS2:ETV1 or TMPRSS2:ERG fusion are indicated in the final column.

TABLE 2 TMPRSS2:ETS family member gene fusion assays TMPRSS2:ETS RLM-family QPCR RACE QPCR QPCR RT-PCR member Case Sample Expressionsequencing TMPRSS2:ETV1 TMPRSS2:ERG sequencing fusion 1 MET26- ETV1* + +− + LN 1 MET26- ETV1* + − + RP 2 MET28-B ERG − + + 2 MET28- ERG − + +PTLN 2 MET28- ERG − + + 41 2 MET28- ERG + − + + LN 3 MET16- ERG − + + 443 MET16- ERG − + + 47 4 MET3 ERG* − + + 5 MET18- ERG* − + + + 23 6 PCA1ERG* + − + + + 7 PCA2 ERG* − + + + 8 PCA3 ERG* − + + + 9 PCA4 ERG* +− + + 10 PCA5 ERG* + − + + 11 PCA6 ERG* − + + 12 PCA7 ERG* + − + + 13PCA8 ERG* − + + 14 PCA9 ERG* − + + 15 PCA10 ERG* − + + 16 PCA11 ERG*− + + 17 PCA12 ERG* − + + 18 PCA13 ERG* − + + 19 PCA14 ERG* − + + 20PCA15 ERG* − + + 21 PCA16 ETV1* − − − − 22 PCA17 ERG* − − − − 23 MET30-− − − − LN 24 MET17- − − − − 12 25 MET20- − − − − 76 26 MET22- − − − −61 27 MET5-7 − − − − 28 PCA18 − − − − 29 PCA19 − − − − 30 PCA20 − − − −31 PCA21 − − − − 32 PCA22 − − − − 33 PCA23 − − − − 34 PCA24 − − − − 35PCA25 − − − − 36 PCA26 − − − − 37 PCA27 − − − − 38 PCA28 − − − − 39PCA29 − − − − 40 PCA30 − − − − 41 PCA31 − − − − 42 PCA32 − − − − CellVCap ERG + − + + line Cell DUCaP ERG − + + + line Cell LnCaP ETV1 − − −− line Cell DU145 − − − − line Cell PC3 − − − − line Cell RWPE − − − −lineTable 3. Cancer Outlier Profile Analysis (COPA). Genes that are known toundergo causal mutations in cancer that had an outlier profile in thetop 10 of a study in Oncomine are shown. “X”, signifies literatureevidence for acquired pathognomonic translocation.

“XX” signifies literature evidence for the specific translocation aswell as that the samples in the specific study were characterized forthat translocation. “Y” signifies consistent with known amplification.“**” signifies ERG and ETV1 outlier profiles in prostate cancer.

TABLE 3 Rank % Score Study Cancer Reference Gene Evidence 1 90 21.9346Bittner et al. Melanoma Nature CDH1 406, 536 (2000) 1 95 20.056 Valk etal. Leukemia Nature RUNX1T1 XX 406, 536 (2000) 1 95 15.4462 Vasselli etal. Renal PNAS PRO1073 X (12) USA 100, 6958 (2003) 1 95 14.2008 Segal etal. Sarcoma J Clin MYH11 Oncol 21, 1775 (2003) 1 90 12.9581 Ross et al.Leukemia Blood PBX1 XX 102, 2951 (2003) 1 95 10.03795 Lapointe etProstate PNAS ETV1 ** al. USA 101, 811 (2004) 1 90 9.1163 Prostate ETV1** 1 90 7.4557 Tian et al. Myeloma N Engl J WHSC1 X (16) Med 349, 2483(2003) 1 75 5.4071 Dhanasekaran Prostate Faseb J ERG ** et al. 19, 243(2005) 1 75 5.2067 Wang et al. Breast Lancet FOXO3A 365, 671 (2005) 1 754.3628 Welsh et al. Prostate Cancer ERG ** Res 61, 5974 (2001) 1 754.3425 Zhan et al. Myeloma Blood 99, CCND1 X (21) 1745 (2002) 1 75 3.724Cheok et al. Leukemia Nat Genet PCSK7 34, 85 (May, 2003) 1 75 3.4414Lapointe et Prostate PNAS ERG ** al. USA 101, 811 (2004) 1 75 3.3875Dhanasekaran Prostate Nature ERG ** et al. 412, 822 (2001) 1 75 2.5913Wigle et al. Lung Cancer IGH@ Res 62, 3005 (2002) 2 90 12.7953 Ross etal. Leukemia Blood HOXA9 102, 2951 (2003) 2 95 9.2916 Golub et al.Leukemia Science TRA@ 286, 531 (1999) 2 95 9.2916 Golub et al. LeukemiaScience TRD@ 286, 531 (1999) 2 90 8.2292 Cheok et al. Leukemia Nat GenetSSX2 34, 85 (May, 2003) 2 90 6.7029 Prostate ERG ** 3 95 13.3478 Zhan etal. Myeloma Blood 99, FGFR3 X (21) 1745 (2002) 3 95 10.2267 Cheok et al.Leukemia Nat Genet ARHGAP26 34, 85 (May, 2003) 3 90 5.9174 Prostate REL3 75 2.6162 Rosenwald et Lymphoma Cancer TCL1A al. Cell 3, 185 (2003) 375 2.036 Sotiriou et al. Breast PNAS RAD51L1 USA 100, 10393 (2003) 4 758.4985 Bittner et al. Melanoma Nature TP53 406, 536 (2000) 4 90 5.4881Golub et al. Leukemia Science LCK 286, 531 (1999) 4 75 2.5728 Huang etal. Breast Lancet ERBB2 Y(29) 361, 1590 (2003) 4 75 2.0229 Schwartz etOvarian Cancer IGL@ al. Res 62, 4722 (2002) 6 90 17.3733 Ferrando etLeukemia Cancer ZBTB16 al. Cell 1, 75 (2002) 6 95 9.1267 Chen et al.Gastric Mol Biol FGFR2 Cell 14, 3208 (2003) 6 90 6.6079 Sotiriou et al.Breast PNAS ERBB2 Y(29) USA 100, 10393 (2003) 6 75 5.7213 LaTulippe etal. Prostate Cancer NF1 Res 62, 4499 (2002) 6 75 5.2752 Jain et al.Endocrine Cancer PHOX2B Res 64, 3907 (2004) 6 90 4.8383 Lapointe et al.Prostate PNAS LAF4 USA 101, 811 (2004) 6 90 4.1779 Alizadeh et al.Lymphoma Nature IRTA1 403, 503 (2000) 6 90 3.6325 Rosenwald et al.Lymphoma N Engl J IRTA1 Med 346, 1937 (2002) 6 75 1.85865 Chen et al.Liver Mol Biol HMGA1 Cell 13, 1929 (2002) 7 95 4.7561 Alon et al. ColonProc Natl NONO Acad Sci USA 96, 6745 (1999) 7 75 1.8133 Chen et al.Liver Mol Biol GPC3 Cell 13, 1929 (2002) 8 90 4.7068 Lacayo et al.Leukemia Blood EVI1 104, 2646 (2004) 8 90 4.7068 Lacayo et al. LeukemiaBlood MDS1 104, 2646 (2004) 9 95 17.1698 Glinsky et al. Prostate J ClinETV1 ** Invest 113, 913 (2004) 9 90 15.3889 Ferrando et al. LeukemiaFerrando MN1 et al., Cancer Cell 1, 75 (2002) 9 90 6.60865 Nielsen etal. Sarcoma Lancet SSX1 X (42) 359, 1301 (2002) 9 90 4.4875 Lapointe etal. Prostate PNAS CHEK2 USA 101, 811 (2004) 9 75 2.2218 Yu et al.Prostate J Clin ERG ** Oncol 22, 2790 (2004) 10 95 10.6036 Segal et al.Sarcoma Segal et KIT al., J Clin Oncol 21, 1775 (2003)Table 4. Oligonucleotide primers used in this study. For all primers,the gene, bases and exons (according to alignment of the referencesequences described in the text with the May 2004 assembly of the humangenome using the UCSC Genome Browser) are listed. Forward primers areindicated with “f” and reverse primers with “r”.

TABLE 4 Gene Bases Exon(s) Primer Sequence 5′ to 3′ SEQ ID NO ETV1 193-2 Exon 2- AACAGAGATCTGGCTCATGATTCA 1 216 3_f ETV1 268- 3 Exon 2-CTTCTGCAAGCCATGTTTCCTGTA 2 245 3_r ETV1 248- 3-4 Exon 3-AGGAAACATGGCTTGCAGAAGCTC 3 271 4_f ETV1 305- 4 Exon 3-TCTGGTACAAACTGCTCATCATTGTC 4 280 4_r ETV1 269- 4 Exon 4-CTCAGGTACCTGACAATGATGAGCAG 5 294 5_f ETV1 374- 5 Exon 4-CATGGACTGTGGGGTTCTTTCTTG 6 351 5_r ETV1 404- 5 Exon 5-AACAGCCCTTTAAATTCAGCTATGGA 7 429 6_f ETV1 492- 6 Exon 5-GGAGGGCCTCATTCCCACTTG 8 472 6_r ETV1 624- 6-7 Exon 6-CTACCCCATGGACCACAGATTT 9 645 7f_ ETV1 771- 7 Exon 6-CTTAAAGCCTTGTGGTGGGAAG 10 750 7_r ERG 574- 5-6 Exon 5-CGCAGAGTTATCGTGCCAGCAGAT 11 597 6_f ERG 659- 6 Exon 5-CCATATTCTTTCACCGCCCACTCC 12 636 6_r NA NA NA GeneraceCGACTGGAGCACGAGGACACTGA 13 r 5′_f ETVI 374- 5 Exon 4-CATGGACTGTGGGGTTCTTTCTTG 14 351 5_r ERG 284- 4 ExonGGCGTTCCGTAGGCACACTCAA 15 263 4a_r ERG 396- 4 Exon CCTGGCTGGGGGTTGAGACA16 377 4b_r TMPRS −4- 1 TMPRSS TAGGCGCGAGCTAAGCAGGAG 17 S2 17 2:ERG_fERG 276- 4 TMPRSS GTAGGCACACTCAAACAACGACTGG 18 252 2:ERG_r TMPRS 1-19 1TMPRSS CGCGAGCTAAGCAGGAGGC 19 S2 2:ETV1_f ETV1 339- 4-5 TMPRSSCAGGCCATGAAAAGCCAAACTT 20 318 2:ETV1_r

Example 2 ETV4 Gene Fusions A. Materials and Methods ETS FamilyExpression in Profiling Studies

To investigate the expression of ETS family members in prostate cancer,two prostate cancer profiling studies were utilized (Lapointe et al.,Proc Natl Acad Sci USA 2004; 101:811-6 and Tomlins et al., Science 2005;310:644-8) present in the Oncomine database (Rhodes et al., Neoplasia2004; 6:1-6). Genes with an ETS domain were identified by the Interprofilter ‘Ets’ (Interpro ID: IPR000418). Heatmap representations weregenerated in Oncomine using the ‘median-center per gene’ option, and thecolor contrast was set to accentuate ERG and ETV1 differentialexpression.

Samples

Prostate cancer tissues (PCA1-5) were from the radical prostatectomyseries at the University of Michigan, which is part of the University ofMichigan Prostate Cancer Specialized Program of Research Excellence(S.P.O.R.E.) Tissue Core. All samples were collected with informedconsent of the patients and prior institutional review board approval.Total RNA was isolated with Trizol (Invitrogen, Carlsbad, Calif.)according to the manufacturer's instructions. A commercially availablepool of benign prostate tissue total RNA (CPP, Clontech, Mountain View,Calif.) was also used.

Quantitative PCR (QPCR)

QPCR was performed using SYBR Green dye on an Applied Biosystems 7300Real Time PCR system (Applied Biosystems, Foster City, Calif.) asdescribed (Tomlins et al., supra). The amount of each target generelative to the housekeeping gene glyceraldehyde-3-phosphatedehydrogenase (GAPDH) for each sample was reported. The relative amountof the target gene was calibrated to the relative amount from the poolof benign prostate tissue (CPP). All oligonucleotide primers weresynthesized by Integrated DNA Technologies (Coralville, Iowa). GAPDHprimers were as described (Vandesompele et al., Genome Biol 2002;3:RESEARCH0034). Primers for exons of ETV4 were as follows (listed 5′ to3′): ETV4_exon2-f: CCGGATGGAGCGGAGGATGA (SEQ ID NO:21), ETV4_exon2-r:CGGGCGATTTGCTGCTGAAG (SEQ ID NO:22), ETV4_exon3-f: GCCGCCCCTCGACTCTGAA(SEQ ID NO:23), ETV4_exon4-r: GAGCCACGTCTCCTGGAAGTGACT (SEQ ID NO:24),ETV4_exon11-f: CTGGCCGGTTCTTCTGGATGC (SEQ ID NO:25), ETV4_exon12-r:CGGGCCGGGGAATGGAGT (SEQ ID NO:26), ETV4_(—)3′UTR-f:CCTGGAGGGTACCGGTTTGTCA (SEQ ID NO:27), ETV4_(—)3′UTR-r:CCGCCTGCCTCTGGGAACAC (SEQ ID NO:28). Exons were numbered by alignment ofthe RefSeq for ETV4 (NM_(—)001986.1) with the May 2004 freeze of thehuman genome using the UCSC Genome Browser. For QPCR confirmation ofTMPRSS2:ETV4 fusion transcripts, TMPRSS2:ETV4α-f(AAATAAGTTTGTAAGAGGAGCCTCAGCATC (SEQ ID NO:29)) and TMPRSS2:ETV4b-f(ATCGTAAAGAGCTTTTCTCCCCGC (SEQ ID NO:30)), which detects bothTMPRSS2:ETV4a and TMPRSS2; ETV4b transcripts, were used withETV4_exon4-r.

RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE)

RLM-RACE was performed using the GeneRacer RLM-RACE kit (Invitrogen),according to the manufacturer's instructions as described (Tomlins etal., supra). To obtain the 5′ end of ETV4, first-strand cDNA from PCA5was amplified using the GeneRacer 5′ Primer and ETV4_exon4-r orETV4_exon7-r (GAAAGGGCTGTAGGGGCGACTGT (SEQ ID NO:31)). Products werecloned and sequenced as described (Tomlins et al., supra). Equivalent 5′ends of the TMPRSS2:ETV4 transcripts were obtained from both primerpairs.

Fluorescence In Situ Hybridization (FISH)

Formalin-fixed paraffin-embedded (FFPE) tissue sections were used forinterphase FISH. Deparaffinized tissue was treated with 0.2 M HCl for 10min, 2×SSC for 10 min at 80° C. and digested with Proteinase K(Invitrogen) for 10 min. The tissues and BAC probes were co-denaturedfor 5 min at 94° C. and hybridized overnight at 37° C.Post-hybridization washing was with 2×SSC with 0.1% Tween-20 for 5 minand fluorescent detection was performed using anti-digoxigeninconjugated to fluorescein (Roche Applied Science, Indianapolis, Ind.)and streptavidin conjugated to Alexa Fluor 594 (Invitrogen). Slides werecounterstained and mounted in ProLong Gold Antifade Reagent with DAPI(Invitrogen). Slides were examined using a Leica DMRA fluorescencemicroscope (Leica, Deerfield, Ill.) and imaged with a CCD camera usingthe CytoVision software system (Applied Imaging, Santa Clara, Calif.).

All BACs were obtained from the BACPAC Resource Center (Oakland, Calif.)and probe locations were verified by hybridization to metaphase spreadsof normal peripheral lymphocytes. For detection of TMPRSS2:ETV4 fusion,RP11-35C4 (5′ to TMPRSS2) was used with multiple BACs located 3′ to ETV4(distal to ETV4 to proximal: RP11-266124, RP11-242D8, and RP11-100E5).For detection of ETV4 rearrangements, RP11-436J4 (5′ to ETV4) was usedwith the multiple BACs 3′ to ETV4. For each hybridization, areas ofcancerous cells were identified by a pathologist and 100 cells werecounted per sample. The reported cell count for TMPRSS2:ETV4 fusionsused RP11-242D8 and similar results were obtained with all 3′ ETV4 BACs.To exclude additional rearrangements in PCA5, FISH was performed withtwo probes 3′ to ETV4 (RP11-266124 and RP11-242D8), ERG split signalprobes (RP11-95I21 and RP11-476D17) and TMPRSS2:ETV1 fusion probes(RP11-35C4 and RP11-124L22). BAC DNA was isolated using a QIAFilter MaxiPrep kit (Qiagen, Valencia, Calif.) and probes were synthesized usingdigoxigenin- or biotin-nick translation mixes (Roche Applied Science).

B. Results

The initial COPA screen led to the characterization of TMPRSS2 fusionswith ERG or ETV1 (Example 1). It was further contemplated that prostatecancers negative for these gene fusions harbor rearrangements involvingother ETS family members. By interrogating the expression of all ETSfamily members monitored in prostate cancer profiling studies from theOncomine database (Rhodes et al., supra), marked over-expression of theETS family member ETV4 was identified in a single prostate cancer casefrom each of two studies—one profiling grossly dissected tissues(Lapointe et al., supra) (FIG. 7A) and the other profiling laser capturemicrodissected (LCM) tissues (FIG. 7B). As these cases did notover-express ERG or ETV1, and no benign prostate tissues showedover-expression, it was contemplated that fusion with TMPRSS2 wasresponsible for the over-expression of ETV4 in these cases. AlthoughELF3 was also over-expressed in a fraction of prostate cancer cases, inboth studies normal prostate tissue samples also showed marked ELF3over-expression, indicating that a gene fusion driving expression inboth benign and cancerous tissue is unlikely. Thus, the ETV4over-expressing case (designated here as PCA5) was further analyzed.

Total RNA was isolated from PCA5 and exon-walking quantitative PCR wasused (QPCR) to confirm the over-expression of ETV4. QPCR demonstratedthat exons 3′ to exon 2 of ETV4 were markedly over-expressed in thiscase compared to pooled benign prostate tissue (CPP) (˜900 fold) andprostate cancers that did not over-express ETV4 and were eitherTMPRSS2:ERG positive (PCA1-2) or negative (PCA3-4) (FIG. 8A). However, adramatic decrease (>99%) in the expression of exon 2 of ETV4 relative todistal regions in PCA5 was observed, indicating a possible fusion withTMPRSS2, as observed previously in TMPRSS2:ERG and TMPRSS2:ETV1 positivecases (Tomlins et al., supra).

To identify the 5′ end of the ETV4 transcript in PCA5, RNA-ligasemediated rapid amplification of cDNA ends (RLM-RACE) was performed usinga reverse primer in exon 7. RLM-RACE revealed two transcripts, eachcontaining 5′ ends consisting of sequence located approximately 8 kbupstream of TMPRSS2 fused to sequence from ETV4 (FIG. 8B). Specifically,the 5′ end of TMPRSS2:ETV4a has 47 base pairs from this region upstreamof TMPRSS2, while the 5′ end of TMPRSS2:ETV4b has the same terminal 13base pairs. These 5′ ends of both transcripts were fused to the samecontiguous stretch consisting of the 9 base pairs of the intronimmediately 5′ to exon 3 of ETV4 and the reported reference sequence ofexons 3 through the reverse primer in exon 7 of ETV4.

The existence of both transcripts in PCA5 and their absence in CPP andPCA1-4 was confirmed using QPCR. To further exclude the presence offusion transcripts involving known exons from TMPRSS2, QPCR wasperformed using a forward primer in exon 1 of TMPRSS2 and the ETV4 exon4 reverse primer, and as expected, no product was detected in CPP orPCA1-5.

Whether other prostate cancers with ETV4 dysregulation might containTMPRSS2:ETV4 fusion transcripts structurally more similar to TMPRSS2:ERGand TMPRSS2:ETV1 transcripts (which involve known exons from TMPRSS2) isunknown. The TMPRSS2:ETV4 fusions reported here do not contain the wellcharacterized AREs immediately upstream of TMPRSS2. However, evidenceexists for androgen responsive enhancers located upstream of the TMPRSS2sequences present in the TMPRSS2:ETV4 transcripts described here(Rabbitts, Nature 1994; 372:143-9). Nevertheless, the markedover-expression of only ETV4 exons involved in the fusion transcriptstrongly suggests that the gene fusion is responsible for thedysregulation of ETV4. Together, the structure of the TMPRSS2:ETV4fusion transcripts supports the conclusion that the regulatory elementsupstream of TMPRSS2, rather than transcribed TMPRSS2 sequences, drivethe dysregulation of ETS family members.

To confirm the fusion of the genomic loci surrounding TMPRSS2 (21q22)and ETV4 (17q21) as demonstrated by RLM-RACE and QPCR, interphasefluorescence in situ hybridization (FISH) was used. Using probes 5′ toTMPRSS2 and 3′ to ETV4, fusion of TMPRSS2 and ETV4 loci was observed in65% of cancerous cells from PCA5 (FIG. 8D). As further confirmation ofthe rearrangement of ETV4, using probes 5′ and 3′ to ETV4, 64% ofcancerous cells from PCA5 showed split signals. FISH was also performedon PCA5 using two probes 3′ to ETV4, ERG split signal probes andTMPRSS2:ETV1 fusion probes to exclude additional rearrangements, withnegative results obtained for each hybridization.

Taken together, the results highlight the use of carefully examiningoutlier profiles in tumor gene expression data, as most analyticalmethods discount profiles that do not show consistent deregulation(Eisen et al., Proc Natl Acad Sci USA 1998; 95:14863-8; Golub et al.,Science 1999; 286:531-7; Tusher et al., Proc Natl Acad Sci USA 2001;98:5116-21) and would thus fail to identify ETV4 in prostate cancer,which appears rare (2 of 98 cases). Combined with the identification ofTMPRSS2:ERG and TMPRSS2:ETV1 fusions, the results presented here showthat dysregulation of ETS family members mediated by subversion of AREsor enhancers upstream of TMPRSS2 is a hallmark of prostatetumorigenesis.

Example 3 Detection of Gene Fusion RNA

This example describes target capture, amplification and qualitativedetection of RNA (IVT) containing the sequences of the four gene fusionsin four separate qualitative assays: TMPRSS2:ETV1a, TMPRSS2:ETV1b,TMPRSS2:ERGa and TMPRSS2:ERGb with APTIMA formulation reagents and HPAdetection each spiked with the appropriate target specificoligonucleotides, primers and probes. Table 5 shows sequences ofoligonucleotides used in the assay.

TABLE 5 SEQ ID Gene Fusion Sequence (5′ to NO TMPRSS2 exon1/AAAAAAAAAAAAAAAAAAAAAAAAAAAAAATTTCUCGAUUCGUCCUCCG 59 Target CaptureTMPRSS2 exon1/ AAAAAAAAAAAAAAAAAAAAAAAAAAAAAATTTAUCCGCGCUCGAUUCGUC 60Target Capture TMPRSS2 exon1/ GAGGGCGAGGGGCGGGGAGCGCC 61 Non-T7 TMPRSS2exon2/ CCTATCATTACTCGATGCTGTTGATAACAGC 62 Non-T7 ETV1a/b exon4/T7AATTTAATACGACTCACTATAGGGAGAAACTTTCAGCCTGATA 63 ERGb exon2/T7AATTTAATACGACTCACTATAGGGAGACTCTGTGAGTCATTTGTCTTGCTT 64 ERGa exon4/T7AATTTAATACGACTCACTATAGGGAGAGCACACTCAAACAACGACTG 65 TMPRSS2exon1:GCGCGGCAG-CUCAGGUACCUGAC 66 ETV1a Junction/AE TMPRSS2exon2:GCUUUGAACUCA-CUCAGGUACCUGAC 67 ETV1b Junction/AE TMPRSS2exon1:GAGCGCGGCAG-GAAGCCUUAUCAGUUG 68 ERGa Junction/AE TMPRSS2exon1:GAGCGCGGCAG-GUUAUUCCAGGAUCUUU 69 ERGb Junction/AE

A. Materials and Methods RNA Target Capture

Lysis buffer contained 15 mM sodium phosphate monobasic monohydrate, 15mM sodium phosphate dibasic anhydrous, 1.0 mM EDTA disodium dihydrate,1.0 mM EGTA free acid, and 110 mM lithium lauryl sulfate, pH 6.7. Targetcapture reagent contained 250 mM HEPES, 310 mM lithium hydroxide, 1.88 Mlithium chloride, 100 mM EDTA free acid, at pH 6.4, and 250 μg/ml 1micron magnetic particles SERA-MAG MG-CM Carboxylate Modified (Seradyn,Inc., Indianapolis, Ind.) having dT)₁₄ oligomers covalently boundthereto. Wash solution contained 10 mM HEPES, 6.5 mM sodium hydroxide, 1mM EDTA, 0.3% (v/v) ethanol, 0.02% (w/v) methyl paraben, 0.01% (w/v)propyl paraben, 150 mM sodium chloride, 0.1% (w/v) lauryl sulfate,sodium (SDS), at pH 7.5.

RNA Amplification & Detection

Amplification reagent was a lyophilized form of a 3.6 mL solutioncontaining 26.7 mM rATP, 5.0 mM rCTP, 33.3 mM rGTP and 5.0 mM rUTP, 125mM HEPES, 8% (w/v) trehalose dihydrate, 1.33 mM dATP, 1.33 mM dCTP, 1.33mM dGTP and 1.33 mM dTTP, at pH 7.5. The Amplification reagent wasreconstituted in 9.7 mL of the amplification reagent reconstitutionsolution (see below). Before use, 15 pmol each of primer oligomers wasadded. Amplification reagent reconstitution solution contained 0.4%(v/v) ethanol, 0.10% (w/v) methyl paraben, 0.02% (w/v) propyl paraben,33 mM KCl, 30.6 mM MgCl₂, 0.003% phenol red. Enzyme reagent was alyophilized form of a 1.45 mL solution containing 20 mM HEPES, 125 mMN-acetyl-L-cysteine, 0.1 mM EDTA disodium dihydrate, 0.2% (v/v) TRITON7X-100 detergent, 0.2 M trehalose dihydrate, 0.90 RTU/mL Moloney murineleukemia virus (MMLV) reverse transcriptase, and 0.20 U/mL T7 RNApolymerase, at pH 7.0. One unit (RTU) of activity is defined as thesynthesis and release of 5.75 fmol cDNA in 15 minutes at 37° C. for MMLVreverse transcriptase, and for T7 RNA polymerase, one unit (U) ofactivity is defined as the production of 5.0 fmol RNA transcript in 20minutes at 37° C. Enzyme reagent was reconstituted in 3.6 mL of theenzyme reagent reconstitution solution (see below). Enzyme reagentreconstitution solution contained 50 mM HEPES, 1 mM EDTA, 10% (v/v)TRITON7 X-100, 120 mM potassium chloride, 20% (v/v) glycerol anhydrous,at pH 7.0. Hybridization reagent contained 100 mM succinic acid freeacid, 2% (w/v) lithium lauryl sulfate, 100 mM lithium hydroxide, 15 mMaldrithiol-2, 1.2 M lithium chloride, 20 mM EDTA free acid, 3.0% (v/v)ethanol, at pH 4.7. Selection reagent contained 600 mM boric acid, 182.5mM sodium hydroxide, 1% (v/v) TRITON7 X-100, at pH 8.5. The detectionreagents comprised detect reagent I, which contained 1 mM nitric acidand 32 mM hydrogen peroxide, and detect reagent II, which contained 1.5M sodium hydroxide.

B. Assay Protocol Target Capture

-   -   1. Prepare samples by making dilutions of IVT stock solution        into STM at indicated copy levels for 400 μL sample per reaction        tube.    -   2. Using the repeat pipettor, add 100 μL of the TCR with the TCO        to the appropriate reaction tube.    -   3. Using the micropipettor, add 400 μL of each sample to the        properly labeled.    -   4. Cover the tubes with the sealing card(s) and shake the rack        gently by hand. Do not vortex. Incubate the rack at 62°±1° C. in        a water bath for 30±5 minutes.    -   5. Remove the rack from the water bath and blot bottoms of tubes        dry on absorbent material.    -   6. Ensure the sealing cards are firmly seated. If necessary,        replace with new sealing cards and seal tightly.    -   7. Without removing sealing cards, incubate the rack at room        temperature for 30±5 minutes.    -   8. Place the rack on the TCS magnetic base for 5 to 10 minutes.    -   9. Prime the dispense station pump lines by pumping APTIMA Wash        Solution through the dispense manifold. Pump enough liquid        through the system so that there are no air bubbles in the line        and all 10 nozzles are delivering a steady stream of liquid.    -   10. Turn on the vacuum pump and disconnect the aspiration        manifold at the first connector between the aspiration manifold        and the trap bottle. Ensure that the vacuum gauge reads greater        than 25 in. Hg. It may take 15 seconds to achieve this reading.        Reconnect the manifold, and ensure the vacuum gauge is between 7        and 12 in. Hg. Leave the vacuum pump on until all target capture        steps are completed.    -   11. Firmly attach the aspiration manifold to the first set of        tips. Aspirate all liquid by lowering the tips into the first        TTU until the tips come into brief contact with the bottoms of        the tubes. Do not hold the tips in contact with the bottoms of        the tubes.    -   12. After the aspiration is complete, eject the tips into their        original tip cassette. Repeat the aspiration steps for the        remaining TTUs, using a dedicated tip for each specimen.    -   13. Place the dispense manifold over each TTU and, using the        dispense station pump, deliver 1.0 mL of APTIMA Wash Solution        into each tube of the TTU.    -   14. Cover tubes with a sealing card and remove the rack from the        TCS. Vortex once on the multi-tube vortex mixer.    -   15. Place rack on the TCS magnetic base for 5 to 10 minutes.    -   16. Aspirate all liquid as in steps 13 and 14.    -   17. After the final aspiration, remove the rack from the TCS        base and visually inspect the tubes to ensure that all liquid        has been aspirated. If any liquid is visible, place the rack        back onto the TCS base for 2 minutes, and repeat the aspiration        for that TTU using the same tips used previously for each        specimen.

Primer Annealing and Amplification

-   -   1. Using the repeat pipettor, add 75 μL of the reconstituted        Amplification Reagent containing the analyte specific primers to        each reaction tube. All reaction mixtures in the rack should now        be red in color.    -   2. Using the repeat pipettor, add 200 μL of Oil Reagent.    -   3. Cover the tubes with a sealing card and vortex on the        multi-tube vortex mixer.    -   4. Incubate the rack in a water bath at 62°±1° C. for 10±5        minutes.    -   5. Transfer the rack into a water bath at 42°±1° C. for 5±2        minutes.    -   6. With the rack in the water bath, carefully remove the sealing        card and, using the repeat pipettor, add 25 μL of the        reconstituted Enzyme Reagent to each of the reaction mixtures.        All reactions should now be orange in color.    -   7. Immediately cover the tubes with a fresh sealing card, remove        from the water bath, and mix the reactions by gently shaking the        rack by hand.    -   8. Incubate the rack at 42°±1° C. for 60±15 minutes.

Hybridization

-   -   1. Remove the rack from the pre-amplification water bath and        transfer to the post-amplification area. Add 100 μL of the        reconstituted Probe Reagent with analyte specific probe, using        the repeat pipettor. All reaction mixtures should now be yellow        in color.    -   2. Cover tubes with a sealing card and vortex for 10 seconds on        the multi-tube vortex mixer.    -   2. Incubate the rack in a 62°±1° C. water bath for 20±5 minutes.    -   3. Remove the rack from the water bath and incubate at room        temperature for 5±1 minutes

Selection

-   -   1. Using the repeat pipettor, add 250 μL of Selection Reagent to        each tube. All reactions should now be red in color.    -   2. Cover tubes with a sealing card, vortex for 10 seconds or        until the color is uniform, and incubate the rack in a water        bath at 62°±1° C. for 10±1 minutes.    -   3. Remove the rack from the water bath. Incubate the rack at        room temperature for 15±3 minutes.

Reading the TTUs

-   -   1. Ensure there are sufficient volumes of Auto Detection Regents        I and II to complete the tests.    -   2. Prepare the LEADER Luminometer by placing one empty TTU in        cassette position number 1 and perform the WASH protocol.    -   3. Load the TTUs into the luminometer and run the HC+ Rev B        protocol.

C. Results

The results are shown in Tables 6-9 for 4 assays with each of theTMPRSS2:ERG and TMPRSS2:ETV1 gene fusion IVTs spiked into TCR.

TABLE 6 TMPRSS2:ETV1a (copies IVT/reaction) RLU 0 4,945 0 4,599 102,185,959 10 2,268,090 10 2,284,908 100 2,270,369 100 2,302,023 1002,272,735 1,000 2,279,627 1,000 2,285,742

TABLE 7 TMPRSS2:ETV1b (copies IVT/reaction) RLU 0 7,743 0 6,622 0 7,3700 6,181 0 7,409 10 7,712 10 7,178 10 7,302 10 8,430 10 8,331 100 774,792100 285,712 100 3,361,878 100 1,349,368 100 2,757,334 1,000 3,647,5021,000 3,790,087 1,000 3,813,812 1,000 3,753,743 1,000 3,667,242

TABLE 8 TMPRSS2:ERGa (copies IVT/reaction) RLU 0 7,938 0 7,505 102,043,379 10 387,408 10 978,457 100 2,332,764 100 2,445,544 1002,530,239

TABLE 9 TMPRSS2:ERGb (copies IVT/reaction) RLU 0 5,978 0 6,284 102,700,069 10 2,768,541 100 2,883,091 100 2,779,233 1,000 2,857,247 1,0002,957,914

Example 4 FISH Assay for Gene Fusions

This Example describes the use of fluorescence in situ hybridization(FISH), to demonstrate that 23 of 29 prostate cancer samples harborrearrangements in ERG or ETV1. Cell line experiments suggest that theandrogen-responsive promoter elements of TMPRSS2 mediate theoverexpression of ETS family members in prostate cancer. These resultshave implications in the development of carcinomas and the moleculardiagnosis and treatment of prostate cancer.

Below is a list of the specific BAC probes used in FISH assays.

Clinical FISH Assay for Testing Aberrations in ETS Family Members byFISH

-   -   Testing ETV1-TMPRSS2 fusion with one probe spanning the ETV1 and        one spanning the TMPRSS2 locus    -   BAC for ETV1: RP11-692L4    -   BAC for TMPRSS2: RP11-121A5, (RP11-120C17, PR11-814F13,        RR11-535H11)    -   Testing ERG translocation with set of probes for c-ERG:t-ERG        break apart:    -   BAC for c-ERG: RP11-24A11    -   BACs for t-ERG: RP11-372O17, RP11-137J13    -   Testing ETV1 deletion/amplification with set of probes, one        spanning the ETV1 locus and one reference probe on chromosome 7:    -   BAC for ETV1: RP11-692L4    -   BAC for reference robe on chromosome 7: A commercial probe on        centromere of chr.    -   Testing ERG deletion/amplification with set of probes, one        spanning the ERG locus and one reference probe on chromosome 21:    -   BAC for ERG: RP11-476D17    -   BACs for reference probe on chromosome 21:*    -   Testing TMPRSS2 deletion/amplification with set of probes, one        spanning the TMPRSS2 locus and one reference probe on chromosome        21:    -   BACs for TMPRSS2: RP11-121A5, (RP11-120C17, PR11-814F13,        RR11-535H11)    -   BACs for reference probe on chromosome 21:* *BACs for reference        probe on chromosome 21: PR11-32L6, RP11-752M23, RP11-1107H21,        RP11-639A7, (RP11-1077M21)

Example 5 TMPRSS2:ERG Fusion Associated Deletions

This example describes the presence of common deletions located betweenERG and TMPRSS2 on chromosome 21q22.2-3 associated with the TMPRSS2:ERGfusion. Associations between disease progression and clinical outcomewere examined using a wide range of human PCA samples, 6 cell lines, and13 xenografts.

A. Materials and Methods Clinical Samples

Prostate samples used for this study were collected under an IRBapproved protocol. All clinically localized PCA samples werecharacterized by one pathologist and assigned a Gleason score toeliminate inter-observer differences in pathology reporting. Clinicallylocalized PCA samples were collected as part of an on-going researchprotocol at the University of Ulm. The hormone refractory samples weretaken from the Rapid Autopsy Program of the University of Michigan.

The FISH experiments were conducted on two PCA outcome arrays, whichwere composed of 897 tissue cores (histospots) from 214 patients. Asummary of the patient demographics is presented in Table 10. Allpatients had undergone radical prostatectomy with pelvic lymphadenectomyat the University of Ulm (Ulm, Germany) between 1989 and 2001.Pre-operative PSA ranged between 1 and 314 ng/ml (mean 36 ng/ml). Meanand maximum follow-up was 3.4 and 8.4 yrs, respectively.

Cell Lines and Xenografts

Androgen independent (PC-3, DU-145, HPV10, and 22Rv1) and androgensensitive (LNCaP) PCA cell lines were purchased from the American TypeCulture Collection (Manassas, Va.) and maintained in their definedmedium. HPV10 was derived from cells from a high-grade PCA (Gleasonscore 4+4=8), which were transformed by transfection with HPV18 DNA(18).22Rv1 is a human PCA epithelial cell line derived from a xenograft thatwas serially propagated in mice after castration-induced regression andrelapse of the parental, androgen-dependent CWR22 xenograft. The VCAPcell line was from a vertebral metastatic lesion as part of the RapidAutopsy program at the University of Michigan.

LuCaP 23.1, 35, 73, 77, 81, 86.2, 92.1, and 105 were derived frompatients with androgen independent hormone-refractory disease PCA. LuCaP49 and 115 are from patients with androgen dependent PCA. LuCaP 58 isderived from an untreated patient with clinically advanced metastaticdisease and LuCaP 96 was established from a prostate derived tumorgrowing in a patient with hormone refractory PCA. LuCaP 49 (establishedfrom an omental mass) and LuCaP 93 are hormone-insensitive (androgenreceptor [AR]-negative) small cell PCAs. These two xenograftsdemonstrate a neuroendocrine phenotype. LuCaP 23.1 is maintained in SCIDmice, and other xenografts are maintained by implanting tumors in maleBALB/c nu/nu mice.

Determining TMPRSS2:ERG Fusion Status Using Interphase FISH

The FISH analysis for the translocation of TMPRSS2:ERG is describedabove and previously (Tomlins, et al., Science 310:644-8 (2005)). Thisbreak apart assay is presented in FIGS. 11 and 14. For analyzing the ERGrearrangement on chromosome 21q22.2, a break apart probe system wasapplied, consisting of the Biotin-14-dCTP labeled BAC clone RP11-24A11(eventually conjugated to produce a red signal) and the Digoxigenin-dUTPlabeled BAC clone RP11-137J13 (eventually conjugated to produce a greensignal), spanning the neighboring centromeric and telomeric region ofthe ERG locus, respectively. All BAC clones were obtained from theBACPAC Resource Center, Children's Hospital Oakland Research Institute(CHORI), Oakland, Calif.

Using this break apart probe system, a nucleus without ERG rearrangementexhibits two pairs of juxtaposed red and green signals. Juxtaposedred-green signals form a yellow fusion signal. A nucleus with an ERGrearrangement shows break apart of one juxtaposed red-green signal pairresulting in a single red and green signal for the translocated alleleand a combined yellow signal for the non-translocated allele in eachcell. Prior to tissue analysis, the integrity and purity of all probeswere verified by hybridization to normal peripheral lymphocyte metaphasespreads. Tissue hybridization, washing, and fluorescence detection wereperformed as described previously (Garraway, et al, Nature 436:117-22(2005); Rubin, et al., Cancer Res. 64:3814-22 (2004)). At least one TMAcore could be evaluated in 59% PCA cases from two TMAs. The technicaldifficulties with this assay included the absence of diagnostic materialto evaluate, weak probe signals, and overlapping cells preventing anaccurate diagnosis. The remainder of the analysis focused on the 118cases of clinically localized PCA that could be evaluated. 15 cases hadcorresponding hormone naïve metastatic lymph node samples that couldalso be evaluated.

The samples were analyzed under a 100× oil immersion objective using anOlympus BX-51 fluorescence microscope equipped with appropriate filters,a CCD (charge-coupled device) camera and the CytoVision FISH imaging andcapturing software (Applied Imaging, San Jose, Calif.). Evaluation ofthe tests was independently performed by two pathologists both withexperience in analyzing interphase FISH experiments. For each case, itwas attempted to score at least 100 nuclei per case. If significantdifferences between the results of both pathologists were found, thecase was refereed by a third pathologist.

Oligonucleotide SNP Array Analysis

Although SNP arrays were intended for genotyping alleles, the SNP arraydata can provide information on Loss-of-Heterozygosity (Lieberfarb, etal., Cancer Res 63:4781-5 (2003); Lin, et al., Bioinformatics 20:1233-40(2004)) and detection of copy number alterations (Zhao, et al., CancerCell 3:483-95 (2003)). Using SNP array analysis, it was possible toidentify and validate amplified genes in various cancers includingmelanoma (MITF) (Garraway, et al., Nature 436:117-22 (2005)) and PCA(TPD52) (Rubin, et al, Cancer Res. 64:3814-22 (2004)).

SNP detection on the 100K array began with a reduction in genomerepresentation. Two aliquots of 250 ng of genomic DNA were digestedseparately with XbaI HindIII. The digested fragments were independentlyligated to an oligonucleotide linker. The resulting products wereamplified using a single PCR primer under conditions in which 200-2000bp PCR fragments were amplified. These fragments represent asub-fraction of the genome. The SNPs tiled on the arrays have beenpre-selected as they lie within these XbaI and HindIII fragments andhave been validated as robustly detected on the arrays. The derivedamplified pools of DNA were then labeled, fragmented further andhybridized to separate HindIII and XbaI oligonucleotide SNP arrays.

Arrays were scanned with a GeneChip Scanner 3000. Genotyping calls andsignal quantification were obtained with GeneChip Operating System 1.1.1and Affymetrix Genotyping Tools 2.0 software. Only arrays withgenotyping call rates exceeding 90% were analyzed further. Raw datafiles were pre-processed and visualized in dChipSNP Lin, et al.,Bioinformatics 20:1233-40 (2004)). In particular, preprocessing includedarray data normalization to a baseline array using a set of invariantprobes and subsequent processing to obtain single intensity values foreach SNP on each sample using a model based (PM/MM) method (Li, et al.,Proc. Nat'l Acad. Sci. USA 98:31-6 (2001)).

Quantitative PCR for TMPRSS2:ERG and TMPRSS2:ETV1 Fusion Transcripts

QPCR was performed using SYBR Green dye (Qiagen) on a DNA engine Opticon2 machine from MJ Research. Total RNA was reverse transcribed into cDNAusing TAQMAN reverse transcription reagents (Applied Biosystems) in thepresence of random Hexamers. All QPCR reactions were performed with SYBRGreen Master Mix (Qiagen). All Oligonucleotide primers were designed atIntegrated DNA Technologies. Primers that were described by Tomlin etal. (Science 310:644-8 (2005)) and are specific for the fusion wereutilized:

TMPRSS2: ERG_f: TAGGCGCGAGCTAAGCAGGAG, (SEQ ID NO: 55) TMPRSS2: ERG_r:GTAGGCACACTCAAACAACGACTGG, (SEQ ID NO: 56) TMPRSS2: ETV1_fCGCGAGCTAAGCAGGAGGC, (SEQ ID NO: 57) TMPRSS2: ETV-1_r:CAGGCCATGAAAAGCCAAACTT. (SEQ ID NO: 58)

GAPDH primers were previously described (Vandesompele, et al, GenomeBiol 3: RESEARCH 0034 (2002)). 10 μMol of forward and reverse primerwere used and procedures were performed according to the manufacturer'srecommended thermocycling conditions. Threshold levels were set duringthe exponential phase of the QPCR reaction using Opticon Monitoranalysis software version 2.02. The amount of each target gene relativeto the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase(GAPDH) for each sample was determined using the comparative thresholdcycle (Ct) method (Applied Biosystems User Bulletin #2). All reactionswere subjected to melt curve analysis and products from selectedexperiments were resolved by electrophoreses on 2% agarose gel.

Statistics

The clinical and pathology parameters were explored for associationswith rearrangement status and with the presence of the deletion.Chi-squared test and Fisher exact test were used appropriately.Kaplan-Meier analysis was used to generate prostate-specific antigenrecurrence free survival curves of the pathology and the genomicalteration parameters. Log-rank test was used to evaluate statisticalsignificance of associations. Patients with prior neo-adjuvant hormoneablation therapy were excluded. All statistics were performed using SPSS13.0 for Windows (SPSS Inc., Chicago, Ill.) with a significance level of0.05.

B. Results

Detection of Deletions on Chromosome 21 Associated with the TMPRSS2:ERGGene Rearrangement

In order to characterize the frequency of the TMPRSS2:ERG rearrangementin PCA, a modified FISH assay from the assay described by Tomlins, etal. (Science 310:644-8 (2005)) was utilized. The original FISH assayused two probes located on ERG at the centromeric 3′ and telomeric 5′ends. The new assay moved the 5′ probe in a telomeric direction (FIG.14). Using a PCA screening tissue microarray (TMA), it was observed thatapproximately 70% of PCA demonstrating TMPRSS2:ERG rearrangement (FIGS.11A and 11B) also showed a loss of the green signal corresponding to thetelomeric 5′ ERG probe (FIGS. 11C and 11D), suggesting that thischromosomal region was deleted. 100K oligonucleotide SNP arrays wereused to characterize the extent of these deletions. By interrogating 30PCA samples, including cell lines, xenografts and hormone naïve andhormone refractory metastatic PCA samples, genomic loss between ERG andTMPRSS2 on chromosome 21q23 was identified (FIG. 12A-C).

The rearrangement status for TMPRSS2:ERG and TMPRSS2:ETV1 was determinedfor these 30 PCA by FISH and/or qPCR (FIG. 12A, gray and light bluebar). Discrete genomic loss was observed in TMPRSS2:ERG rearrangementpositive samples involving an area between TMPRSS2 and the ERG loci forLuCaP 49, LuCaP 93, ULM LN 13, MET6-9, MET18-2, MET24-28, and MET28-27.The extent of these discrete deletions was heterogeneous. More extensivegenomic loss on chromosome 21 including the area between TMPRSS2 and theERG loci was observed in LuCaP 35, LuCaP 86.2, LuCaP 92.1, and MET3-81.The VCaP cell line and the xenograft LuCap 23.1 did not demonstrate lossin this region. For a subset of samples 45% (5 out of 11) the deletionoccurs in proximity of ERG intron 3. For a majority of samples 64% (7out of 11) the deletion ends in proximity of the SNP located on TMPRSS2(the next SNP in the telomeric direction is about 100K bp distant). TheVCaP cell line shows copy number gain along the entire chromosome 21.

For TMPRSS2:ERG rearrangement positive tumors, 71% (5 of 7) hormonerefractory PCA demonstrate a deletion between TMPRSS2 and the ERG lociwhereas deletion was only identified in 25% (1 of 4) hormone naïvemetastatic PCA samples (ULM LN 13). There is significant homogeneity forthe deletion borders with two distinct sub-classes, distinguished by thestart point of the deletion—either at 38.765 Mb or 38.911 Mb. None ofthe standard PCA cell lines (PC-3, LNCaP, DU-145, or CWR22 (22Rv1))demonstrated the TMPRSS2:ERG or TMPRSS2:ETV1 fusion. Several of theLuCap xenografts demonstrate TMPRSS2:ERG fusion with deletion includingLuCaP 49 (established from an omental mass) and LuCaP 93, bothhormone-insensitive (androgen receptor [AR]-negative) small-cell PCAs.

Copy number gain of ERG was observed in a small subset of cases bothwith and without the TMPRSS2:ERG rearrangement. The VCaP cell linederived from a hormone refractory PCA demonstrated significant copynumber gain on chromosome 21 (FIG. 12A-C), which was confirmed by FISH.

TMPRSS2:ERG Rearrangement in Primary Prostate Cancer Samples and HormoneNaïve Lymph Node Metastases

To characterize the frequency and potential clinical significance ofthese observations, 118 clinically localized PCA cases were examined byFISH. The clinical and pathology demographics are presented in Table 10.This cohort of patients is at high risk of disease recurrence asdemonstrated by high tumor grades (Gleason grade), pathology stage, andpre-treatment PSA levels. Using standard tissue sections from thiscohort, where the large areas of the PCA could be studiedmicroscopically, the TMPRSS2:ERG rearrangement was observed to behomogeneous for a given tumor. The TMA experiments confirmed theseobservations. In PCA cases where 3-6 cores were taken from differentareas of the tumor, 100% concordance was observed for TMPRSS2:ERGrearrangement status (i.e. present or absent). It was also observed thatin cases with the TMPRSS2:ERG rearrangement with deletion, the deletionwas observed in all of the TMA cores from the same patient in 97.9%(94/96) of the cases.

TABLE 10 Clinical and Pathological Demographics of 118 Men withClinically Localized Prostate Cancer Treated by Radial Protatectomy*Count Column N % Age <=median 55 50.0% >median 55 50.0% Preoperative PSA<=4 6 8.2% (ng/ml) >4 and <10 13 17.8% >=10 54 74.0% Gleason Score Sum<7 7 6.0% =7 51 43.6% >7 59 50.4% Nuclear Grade 1 — — 2 38 35.5% 3 6964.5% Pathology Stage (pT) PT2 26 22.2% PT3a 34 29.1% PT3b 57 48.7%Surgical Margins status Negative 30 27.8% Positive 78 72.2% Lymph NodeStatus N₀ 52 44.1% (pN) N₁ 56 47.5% N₂ 10 8.5% PSA Recurrence no 3448.6% yes 36 51.4% *Not all data points were available for all 118 cases

The TMPRSS2:ERG rearrangement was identified in 49.2% of the primary PCAsamples and 41.2% in the hormone naïve metastatic LN samples (FIG. 13A).Deletion of the telomeric probe (green signal) (FIG. 1C-D) was observedin 60.3% (35/58) of the primary PCA samples and 42.9% (3/7) of thehormone naïve lymph node tumors with TMPRSS2:ERG rearrangement.

In the 15 cases where there was matched primary and hormone naïve lymphnode tumors, there was 100% concordance for TMPRSS2:ERG rearrangementstatus with 47% (7 of 15) of the pairs demonstrating the rearrangement.Deletion of the telomeric (green signal) probe was concordantly seen in42.9% (3 of 7) of the pairs.

TMPRSS2:ERG Rearrangement Status and Prostate Cancer Progression

The associations between rearrangement status and clinical andpathological parameters were observed (FIG. 13). TMPRSS2:ERGrearrangement with deletion was observed in a higher percentage of PCAcases with advanced tumor stage (pT) (p=0.03) (FIG. 13B), and thepresence of metastatic disease to regional pelvic lymph nodes (pN₀versus pN₁₋₂) (p=0.02). Associations between TMPRSS2:ERG rearrangementwith and without deletion and clinical outcome as determined by prostatespecific antigen (PSA) biochemical failure for 70 patients where followup data was available were also assessed. Gleason grade, tumor stage,nuclear grade and lymph node status were good predictors of PSAbiochemical failure (all p-values <0.0005). A trend was observed at theunivariate level suggesting a PSA recurrence free survival advantage inTMPRSS2:ERG rearranged PCA cases without deletion as determined by theFISH assay.

Example 6 TMPRSS2:ERG Gene Fusion Associated with Lethal Prostate Cancer

In previous studies, the gene fusions of the 5′-untranslated region ofTMPRSS2 (21q22.3) with the ETS transcription factor family members,either ERG (21q22.2), ETV1 (7p21.2) (Tomlins, et al., Science 310:644-8(2005)), or ETV4 (Tomlins, et al., Cancer Res. 66 (7):3396-400 (2006))provide a mechanism for the over expression of the ETS genes in themajority of prostate cancers. Furthermore, the fusion of an androgenregulated gene, TMPRSS2, and an oncogene suggests that diseaseprogression may vary based on these molecular subtypes. The most commonmechanism for gene fusion is loss of about 2.8 megabases of genomic DNAbetween TMPRSS2 and ERG (FIGS. 17A and B). This example describes therisk of metastases or prostate cancer specific death based on thepresence of the common TMPRSS2:ERG gene fusion.

A. Methods

The study population comprises men with early prostate cancer (T1a-b,Nx, M0) diagnosed at the Örebro University Hospital, Sweden, between1977 and 1991 by transurethral resection of the prostate (TURP) ortransvesical adenoma enucleation for symptomatic benign prostatichyperplasia as described by Andren et al. (J. Urol. 175 (4):1337-40(2006)). Baseline evaluation at diagnosis included physical examination,chest radiography, bone scan and skeletal radiography (if needed). Nodalstaging was not carried out. Because this evaluation provided noevidence for distant metastases, patients were followed expectantly andreceived clinical exams, laboratory tests and bone scans every 6 monthsduring the first 2 years after diagnosis and subsequently at 12-monthintervals. Patients, who developed metastases, as determined by bonescan, were treated with androgen deprivation therapy if they exhibitedsymptoms.

The cause of death in the cohort was determined by review of medicalrecords by the study investigators. A validation study regarding causeof death compared to the Swedish Death Register showed greater than 90%concordance, with no systematic under- or over-reporting of any cause ofdeath (Johansson, et al., Lancet 1 (8642):799-803 (1989)). Follow-up ofthe cohort with respect to mortality was 100% and no patients were lostto follow-up through October 2005. The study endpoint was defined asdevelopment of distant metastases or prostate cancer specific death(median follow-up time 9.1 years, maximum 27 years).

All TURP samples were reviewed by one pathologist to confirm a diagnosisof prostate cancer, determine the Gleason score and nuclear grade, andestimate the tumor burden as previously described (J. Urol. 175(4):1337-40 (2006)). A tissue microarray was assembled using a manualarrayer (Rubin, et al., Cancer Epidemiol. Biomarkers Prev. 14 (6):1424-32 (2005)). The frequency of the TMPRSS2:ERG rearrangement inprostate cancer was assessed using a modified florescence in situhybridization (FISH) assay from the assay originally described byTomlins et al (Science 310:644-8 (2005)). The new assay moved the 5′probe approximately 600 kb in a telomeric direction. At least one TMAcore could be evaluated in 92 of the prostate cancer cases.

B. Results

In this population-based cohort of men diagnosed with localized cancer,the frequency of TMPRSS2:ERG fusion was 15.2% (14/92) (FIGS. 17A and B).TMPRSS2:ERG fusion positive tumors were more likely to have a higherGleason score (two-sided P=0.014) (Table 11). To assess the relation offusion status and lethal prostate cancer, cumulative incidenceregression was used. A significant association between the presence ofthe TMPRSS2:ERG gene fusion and metastases or disease specific death(FIG. 17C) with a cumulative incidence ratio (CIR) of 3.6 (P=0.004, 95%confidence interval [CI]=1.5 to 8.9) was observed. When adjusting forGleason Score, the CIR was 2.4 (P=0.07 and 95% CI=0.9 to 6.1). Thepresent invention is not limited to a particular mechanism. Indeed, anunderstanding of the mechanism is not necessary to practice the presentinvention. Nonetheless, it is contemplated that, based on thehomogeneity of the TMPRSS2:ERG gene fusion in cells in a given tumor andits presence only in invasive prostate cancers (compared to ProstaticIntraepithelial Neoplasia), it is contemplated that this is an earlyevent, which might, in part, contribute to the biology behind thephenotype of the Gleason patterns.

TABLE 11 Prognostic Factors for a Cohort of Men Expectantly Managed forLocalized Prostate Cancer Stratified by the TMPRSS2:ERG Gene FusionStatus TMPRSS2:ERG Fusion Status Variable Negative Positive P value* No.of patients 78 14 Age at diagnosis, y 73 (60 to 103) 73 (58 to 90) .683Gleason Score** Gleason Score <7 48 (61.5%)  3 (21.4%) .014 GleasonScore =7 20 (25.6%)  6 (42.9%) Gleason Score >7 10 (12.8%)  5 (35.7%)Pathologic Stage pT1a 28 (35.9%)  2 (14.3%) .112 pT1b 50 (64.1%) 12(85.7%) Nuclear grade*** 1 53 (67.9%)  7 (53.8%) .585 2 18 (23.1%)  4(30.8%) 3  7 (9.0%)  2 (15.4%) Status**** Survived 12 years without 20(25.6%)  1 (7.1%) .016 metastases or cancer death Death due to othercauses 45 (57.7%)  6 (42.9%) within 12 years Distant metastases or death13 (16.7%)  7 (50.0%) due to prostate Cancer *Clinical parameters ofsubjects having the TMPRSS2:ERG fusion and of subjects not having theTMPRSS2:ERG fusion were compared by use of t tests or chi-square testsfor continuous variable and categorical variables, respectively.**Gleason Score is obtained by summing the major and minor Gleasonpatterns. ***For one case nuclear grade was not assessed.****Individuals who lived at least 12 years and have not developedmetastases or died of prostate cancer as of October 2005 are classifiedas long-term survivors. Individuals who lived less than 12 years and didnot develop metastases are classified as short-term survivors.

Example 7 Detection of TMPRSS2:ETS Fusions in the Urine of Patients withProstate Cancer A. Materials and Methods Urine Collection, RNA Isolationand Amplification

Urine samples were obtained from patients following a digital rectalexam before either needle biopsy or radical prostatectomy. Urine wasvoided into urine collection cups containing DNA/RNA preservative(Sierra Diagnostics). For isolation of RNA, a minimum of 30 ml of urinewere centrifuged at 400 rpm for 15 min at 4° C. RNAlater (Ambion) wasadded to the urine sediments and stored at −20° C. until RNA isolation.Total RNA was isolated using a Qiagen RNeasy Micro kit according to themanufacturer's instructions. Total RNA was amplified using an OmniPlexWhole Transcriptome Amplification (WTA) kit (Rubicon Genomics) accordingto the manufacturer's instructions (Tomlins et al., Neoplasia 8:153[2006]). Twenty five nanograms of total RNA were used for WTA librarysynthesis and the cDNA library was subjected to one round of WTA PCRamplification. Amplified product was purified using a QIAquick PCRPurification kit (Qiagen). For cell line proof of concept experiments,the indicated number of VCaP or LNCaP cells was spiked into 1 ml ofsterile urine and the samples were processed as for voided urine.

Quantitative PCR

Quantitative PCR (QPCR) was used to detect ERG, ETV1 and TMPRSS2:ERGtranscripts from WTA amplified cDNA essentially as described (Tomlins etal., Neoplasia 8:153 [2006], Tomlins et al., Science 310:644 [2005],Example 1 above). For each QPCR reaction, 10 ng of WTA amplified cDNAwas used as template. Reactions for ERG, ETV1, PSA and GAPDH used 2×Power SYBR Green Master Mix (Applied Biosystems) and 25 ng of both theforward and reverse primers. Reactions for TMPRSS2:ERGa used 2× TaqmanUniversal PCR Master Mix and a final concentration of 900 nM forward andreverse primers, and 250 nM probe. For the Taqman assay, samples with Ctvalues greater than 38 cycles were considered to show no amplification.For all samples, the amount of ERG and ETV1 were normalized to theamount of GAPDH. Samples with inadequate amplification of PSA,indicating poor recovery of prostate cells in the urine, were excludedfrom further analysis. ERG (exon5_(—)6 forward) and ETV1 (exon6_(—)7)²,GAPDH³, and PSA⁴ primers were as described. The Taqman primers and probe(MGB labeled) specific for TMPRSS2:ERGa are as follows:

TM-ERGa2_MGB-f; CGCGGCAGGAAGCCTTA (SEQ ID NO: 70) TM-ERGa2_MGB-r;TCCGTAGGCACACTCAAACAAC, (SEQ ID NO: 71) TM-ERGa2_MGB-probe;5′-MGB-CAGTTGTGAGTGAGGACC-NFQ-3′ (SEQ ID NO: 72)

Fluorescene In Situ Hybridization (FISH)

Four μm thick formalin-fixed paraffin-embedded (FFPE) sections frommatched needle biopsies were used for interphase fluorescence in situhybridization (FISH), processed and hybridized as described previously(Example 2 and Tomlins et al., Cancer Res 66:3396 [2006]). BAC probes todetect ERG rearrangements, RP11-95I21 (5′ to ERG) and RP11-476D17 (3′ toERG) were prepared as described previously (Tomlins et al., Cancer Res66:3396 [2006]; Tomlins et al., Science 310:644 [2005]; Examples 1 and 2above).

B. Results

This example describes a non-invasive method to detect prostate cancerby the presence of TMPRSS2:ETS fusion transcripts in prostate cancercells shed into the urine after a digital rectal exam. Results are shownin FIG. 33. As a proof of concept, sterile urine spiked with prostatecancer cell lines expressing high levels of ERG and TMPRSS2:ERG (VCaP)or high levels of ETV1 (LNCaP) was used. As shown in FIG. 33A, it waspossible to detect ERG over-expression exclusively in VCaP at 1,600cells and ETV1 over-expression exclusively in LNCaP at 16,000 cells byquantitative PCR (QPCR).

By correlating the number of spiked VCaP and LNCaP cells to GAPDH Ct(threshold cycle) values, it was observed that, in some cases, urineobtained from patients after a digital rectal exam containedinsufficient cell numbers to reliably detect ERG or ETV1over-expression. Thus, total RNA collected from the urine of patientssuspected of having prostate cancer was amplified using OmniPlex WholeTranscriptome Amplification before QPCR analysis. Using this strategy, acohort of 16 patients where urine was obtained after a digital rectalexam before a needle biopsy to detect prostate cancer was assessed.Subsequent assessment of needle biopsies demonstrated that this cohortcontained 4 patients with benign prostates, 1 with high grade prostaticintraepithelial neoplasia (HGPIN) and 11 with prostate cancer. Inaddition, a cohort of 3 patients with prostate cancer where urine wascollected after a digital rectal exam before radical prostatectomy wasassessed.

Cohort characteristics are presented in Table 12. Each urine specimenwas from a unique patient and was assigned an ID. The source of thesample (pre biopsy or radical prostatectomy (RP) is indicated. Thediagnosis following needle biopsy (including benign, high gradeprostatic intraepithelial neoplasia (HGPIN), and prostate cancer (PCa))is indicated. For patients diagnosed as having prostate cancer followingneedle biopsy, major Gleason, minor Gleason, and Gleason sum score areindicated. For all patients, pre biopsy PSA (ng/ml) and age arereported, if available.

TABLE 12 Biopsy Biopsy Biopsy Sample Gleason Gleason Gleason Pre-Biopsysource Diagnosis Major Minor Score PSA (ng/ml) Pre-Biopsy Benign 4.7Pre-Biopsy Benign 8.3 Pre-Biopsy Benign 6.7 Pre-Biopsy Benign 4Pre-Biopsy HGPIN 9.7 Pre-Biopsy Pca 3 4 7 3.3 Pre-Biopsy Pca 3 3 6 5.99Pre-Biopsy Pca 3 3 6 2.8 Pre-Biopsy Pca 3 3 6 5.9 Pre-Biopsy Pca 4 4 810.6 Pre-Biopsy Pca Pre-Biopsy Pca 4 5 9 11.8 Pre-Biopsy Pca 3 4 7 5.5Pre-Biopsy Pca 3 3 6 3.8 Pre-Biopsy Pca 4 5 9 19.3 Pre-BiopsyPca-treated 3 3 6 Pre-RP Pca Pre-RP Pca Pre-RP Pca

From the needle biopsy cohort, 5 patients were identified with markedover-expression of ERG, 1 of which was diagnosed by needle biopsy ashaving HGPIN, while the other 4 were diagnosed as having prostatecancer. From the radical prostatectomy cohort, 1 of 3 patients withprostate cancer were identified as having high ERG expression (FIG.33B). ETV1 over-expression was not detected in any patients from eithercohort. To confirm the expression of TMPRSS2:ERG in the samples whichover-expressed ERG, a TaqMan primer/probe assay designed to specificallyamplify TMPRSS2:ERGa was utilized. This assay robustly amplified productfrom VCaP cells, which express TMPRSS2:ERGa (Tomlins et al., Science310:644 [2005]). In addition, 5 of the 6 urine samples from patientswith prostate cancer that over-expressed ERG also expressed TMPRSS2:ERGa(Ct values 29.8-38.9), while 0 of the 10 samples from patients withoutERG over-expression expressed TMPRSS2:ERGa. As one sample over-expressedERG without expression of TMPRSS2:ERGa, it is likely that this sampleexpresses other isoforms of the fusion transcript, such as TMPRSS2:ERGbor more recently identified fusion transcripts (Soller et al., GenesChromosomes Cancer 45:717 [2006]; Yoshimoto et al., Neoplasia8:465:2006). To confirm that the presence of TMPRSS2:ERG fusiontranscripts indicates the presence of TMPRSS2:ERG positive canceroustissue, fluorescence in situ hybridization (FISH) was performed usingprobes designed to detect ERG rearrangements on matched tissue sectionsfrom representative cases. Matched tissue was obtained from threepatients with detectable TMPRSS2:ERG transcripts in the urine and adiagnosis of cancer, one patient with detectable TMPRSS2:ERG transcriptsin the urine and a diagnosis of high grade PIN, and two patients withoutdetectable TMPRSS2:ERG transcripts and a diagnosis of cancer. As shownin FIG. 33B, both patients diagnosed with cancer but without detectableTMPRSS2:ERG transcripts in their urine did not harbor ERG rearrangementsin cancerous tissue by FISH. All three patients diagnosed with cancerand with detectable TMPRSS2:ERG transcripts in their urine also showedERG rearrangements in cancerous tissue by FISH. Finally, the patientwith a diagnosis of high grade PIN with detectable TMPRSS2:ERG in theirurine did not show ERG rearrangements in high grade PIN tissue. Thisindicates that this patient may have undiagnosed cancer elsewhere in theprostate, resulting in the presence of detectable TMPRSS2:ERGtranscripts in their urine.

Example 8 TMPRSS2 and ETS Family Genes Fusions in Prostate Cancer

This study describes a comprehensive analysis of the frequency for theTMPRSS2 and ETS family genes rearrangements in a screening-based cohortof 111 American men surgically treated for clinically localized prostatecancer.

A. Materials and Methods Study Population, Clinical Data and ProstateSample Collection:

As a source of clinically localized prostate cancers, a tissuemicroarray (TMA) containing—cores representing cancer and benign tissuewas constructed from 111 men who underwent radical prostatectomy at theUniversity of Michigan as the primary monotherapy (i.e., no adjuvant orneoadjuvant hormonal or radiation therapy). The radical prostatectomyseries is part of the University of Michigan Prostate Cancer SpecializedProgram of Research Excellence (SPORE) Tissue Core. Three cores (0.6 mmin diameter) were taken from each representative tissue block toconstruct the TMA. The TMA construction protocol has been described(Kononen et al., Nat. Med. 4:844 [1998]; Rubin et al., Am J surg Pathol26:312 [2002]). Detailed clinical, pathological, and TMA data remaintained on a secure relational database as previously described(Manley et al., Am J. Pathol. 159:837 [2001]).

Assessment of TMPRSS2-ETS Gene Fusion Using an Interphase FluorescenceIn Situ Hybridization Assay

Four μm thick tissue micro array sections were used for interphasefluorescence in situ hybridization (FISH), processed and hybridized asdescribed previously (Tomlins et al., Science 310:644 [2005]; Tomlins etal., Cancer Res 66:3396 [2006]). Slides were examined using an AxioplanImaging Z1 microscope (Carl Zeiss) and imaged with a CCD camera usingthe ISIS software system in Metafer image analysis system (Meta Systems,Altlussheim, Germany). FISH signals were scored manually (100× oilimmersion) by pathologists in morphologically intact and non-overlappingnuclei and a minimum of 30 cells or the maximum numbers of cancer cellsavailable in three cores from a case were recorded. Cases without 30evaluable cells were reported as insufficient hybridization. All BACswere obtained from the BACPAC Resource Center (Oakland, Calif.), andprobe locations were verified by hybridization to metaphase spreads ofnormal peripheral lymphocytes. For detection of TMPRSS2, ERG and ETV4rearrangements we used the following probes: RP11-35C4 (5′ to TMPRSS2)and RP11-120C17 (3′ to TMPRSS2), RP11-95I21 (5′ to ERG) and RP11-476D17(3′ to ERG), and RP11-100E5 (5′ to ETV4) and RP11-436J4 (3′ to ETV4).For detection of TMPSS2-ETV1 fusion, RP11-35C4 (5′ to TMPRSS2) was usedwith RP11-124L22 (3′ to ETV1). BAC DNA was isolated using a QIAFilterMaxi Prep kit (Qiagen, Valencia, Calif.), and probes were synthesizedusing digoxigenin- or biotin-nick translation mixes (Roche AppliedScience, Indianapolis, Ind.). The digoxigenin and biotin labeled probeswere detected using fluorescein conjugated anti-digoxigenin antibodies(Roche Applied Science) and Alexa 594 conjugated streptavidin(Invitrogen, Carlsbad, Calif.), respectively.

A break apart (TMPRSS2, ERG, ETV4) or fusion (TMPRSS2-ETV1) probestrategy was employed to detect rearrangements at the chromosomal level.Normal signal patterns for TMPRSS2, ERG and ETV4 in DAPI stained nucleiwere indicated by two pairs of colocalized green and red signals. Forthese probes, a rearrangement was confirmed by break apart of one of thetwo colocalized signals. For TMPRSS2-ETV1 fusion, two pairs of separatered and green were recorded as normal, while one pair of separate andone pair of colocalized signals was recorded as a rearrangement.

B. Results and Discussion

This example describes a comprehensive analysis outlining the signatureof TMPRSS2 and ETS transcription factor genes rearrangement in a largescreening-based cohort of American men surgically treated for clinicallylocalized prostate cancer. A TMPRSS2 split probe FISH assay approach wasused to detect the overall frequency of gene rearrangement in prostatecancer with known ETS family partners ERG, ETV1, ETV4 and other unknownpartners, as shown in FIG. 34. It was hypothesized that prostate cancersnegative for three known ETS partners (ERG, ETV1 and ETV4) may harborrearrangements involving other ETS family members. The resultsdemonstrate complex molecular signature of TMPRSS and ETS family genesrearrangement in clinically localized prostate cancer (FIGS. 35A and B).Overall TMPRSS2 was rearranged in 65% of evaluable cases, while ERG,ETV1 and ETV4 were rearranged in 55%, 2% and 2% of evaluable cases (FIG.35A). In 40.5% of cases with TMPRSS2 rearrangement, loss of the 3′ probewas observed, consistent with a chromosomal deletion between TMPRSS2 andERG as a mechanism of gene fusion. These results confirm the highfrequency of TMPRSS2:ETS fusions in prostate cancer and confirm previousstudies showing that TMPRSS2:ERG are by far the most common type(Tomlins et al., Science 310:644; Perner et al., Cancer Res 66:3396[2006]; Yoshimoto et al., Neoplasia 8:4665 [2006]; Soller et al., GenesChromosomes Cancer 45:717 [2006]; Wang et al., Cancer Res 66:8347 [2006]and above examples).

Similar results were observed when the cohort was limited to just thosecases where all four probes were evaluable (FIGS. 35A and B). Thisanalysis confirmed that TMPRSS2:ETS rearrangements are mutuallyexclusive, as no cases showed rearrangments of multiple ETS familymembers. This analysis also demonstrates that a single TMPRSS2 assay caneffectively detect almost all ETS rearrangements, as 23 of the 24 caseswith ERG, ETV1 or ETV4 rearrangement were detected by the TMPRSS2 assay.In all 9 cases where the 5′ ERG probe was deleted, deletion of the 3′TMPRSS2 probe was identified.

Furthermore, two cases were identified with break apart of the TMPRSS2probes, indicating a rearrangement, without rearrangement of ERG, ETV1or ETV4 (cases 32 and 36) and cases with TMPRSS2 rearrangement withoutERG rearrangement where ETV1 and/or ETV4 could not be evaluated. Thesecases suggest that TMPRSS2 may be partnering with novel ETS familymembers or unrelated oncogenes in prostate cancer. Together, theseresults suggest that a single TMPRSS2 assay can provide diagnostic andprognostic information in prostate cancer.

Example 9 PSA Gene Fusions

FISH experiments were used to identify cases that show a split signal byFISH for probes located 5′ and 3′ to PSA. The 5′ and 3′ BACs used todetect the PSA split are RP11-510116 and RP11-26P14, respectively. Apartner for the PSA gene fusion has not yet been identified. These sameprobes also pick up a split in the ETS family member SPIB, as it islocated very close to PSA.

Example 10 FL11 Overexpression

FLI1 expression was assayed in different cell samples not harboring aFLI1 gene fusion. The expression of 5′ and 3′ exons of FLI1 was measuredfrom a case with high FLI1 expression. Results are shown in FIG. 18. Nodifference in the 5′ and 3′ transcript abundance was detected. RACE alsodid not indicate a fusion transcript. FLI1 was overexpressed in prostatecancer relative to control samples. Primers for FLi1 amplification, aswell as TaqMan probes, are shown in FIG. 37.

FISH was also used to identify samples that have split signals for FLI1,indicating a rearrangement, but these cases do not have TMPRSS2:FLI1fusion by FISH. BAC probes are shown in Table 13. These cases also havehigh FLI1 expression.

Example 11 Tissue Microarrays

Tissue microarrays were used to assay for the presence of gene fusions.TMAs used included prostate cancer progression array, prostate canceroutcome array, warm autopsy array, prostate cancer screening array, Ergnegative prostate cancer array, and individual prostate cancer cases.The following gene probes were used on tissue microarrays: TMPRSS2-ETV1fusion probes, Erg split probes, TMPRSS2 split probes, ETV1 splitprobes, ETV4 split probes, and FL1 split probes.

In addition, Erg split probes were used on an outcome array. The resultsare as follows: negative cases: 30, positive case: 29, marginalcases: 1. There was a weak association of Erg positive cases with higherGleason score (≧7).

Protein arrays and mass spec were used to identify nuclear interactorsfor ERG2. The results are shown in FIG. 21.

Example 12 Androgen Regulation of Erg Expression

This Example describes the androgen regulation of Erg expression. LNCap(TMPRSS2-ERG-) and VCaP (TMPRSS2-ERG+) cell lines were used. The cellswere contacted with varying amounts of R1881 for 48 hrs. Expression ofErg, PSA (+ control) and beta-tubulin (− control) were assayed. Theresults are shown in FIG. 19. ERG expression was found to be androgendependent in the VCaP, but not the LNCap cells.

Example 13 Peptide Antibody and Aqua Probe Generation

FIGS. 22-25 shows sequences (underlined) of ERG1, ETV1, FLI-1, and ETV4for use in peptide antibody generation and for making aqua probes.Primers are designed by Applied Biosystems for all ETS family members.Expression is monitored in prostate cancer cases, with high expressionbeing an indicator of a possible gene fusion and an indicator for FISH.

Example 14 ETV1 in LnCaP Cells

This Example describes an analysis of the transcriptional response toandrogen in VCaP and LNCaP. In addition to detecting a number oftranscripts differentially expressed in both cell lines were identified,such as PSA, a number of transcripts uniquely dysregulated in VCaP orLNCaP cells were also identified. This analysis identified ETV1 as beingexclusively responsive to androgen in LNCaP cells. Combined with theover-expression of ETV1 in LNCaP cells, FISH was used to interrogate theETV1 loci in LNCaP cells.

A. Materials And Methods Cell Lines

The prostate cancer cell lines LNCaP (originally derived from a lymphnode prostate cancer metastasis) and VCaP (Korenchuk, S. et al., In vivo15, 163-8 (2001)) (originally derived from a vertebral prostate cancermetastasis) were used for this study. For microarray studies, VCaP andLNCaP cells were grown in charcoal-stripped serum containing media for24 hours before treatment for 48 hours with 0.1% ethanol or 1 nM of thesynthetic androgen methyltrienolone (R1881, NEN Life Science Products,Boston, Mass.) dissolved in ethanol. For quantitative PCR (QPCR)studies, cells were grown in charcoal-stripped serum containing mediafor 24 hours, preincubated with 0.1% ethanol, Casodex dissolved inacetone (10 uM, bicalutamide, AstraZeneca Pharmaceuticals, Wilmington,Del.) or flutamide dissolved in ethanol (10 uM, Sigma, St. Louis, Mo.).After 2 hours, 0.1% ethanol or 0.5 nM of R1881 was added and the cellswere harvested after 48 hours. Total RNA was isolated from all sampleswith Trizol (Invitrogen, Carlsbad, Calif.) according to themanufacturer's instructions. RNA integrity was verified by denaturingformaldehyde gel electrophoresis or the Agilent Bioanalyzer 2100(Agilent Technologies, Palo Alto, Calif.).

Microarray Analysis

The cDNA microarrays used for this study were constructed essentially asdescribed, except the array contains 32,448 features. Protocols forprinting and postprocessing of arrays are available on the Internet.cDNA microarray analysis was done essentially as described. Briefly,total RNA from control and R1881 treated VCaP and LNCaP cell lines werereverse transcribed and labeled with cy5 fluorescent dye. Pooled totalRNA from control VCaP or LNCaP samples were reverse transcribed andlabeled with cy3 fluorescent dye for all hybridizations from therespective cell lines. The labeled products were then mixed andhybridized to the cDNA arrays. Images were flagged and normalized usingthe Genepix software package (Axon Instruments Inc., Union City,Calif.). Data were median-centered by arrays and only genes that hadexpression values in at least 80% of the samples were used in theanalysis.

Quantitative PCR (QPCR)

QPCR was performed using SYBR Green dye on an Applied Biosystems 7300Real Time PCR system (Applied Biosystems, Foster City, Calif.) asdescribed (Tomlins et al., Cancer Res 66, 3396-400 (2006); Tomlins etal., Science 310, 644-8 (2005)). The amount of each target gene relativeto the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase(GAPDH) for each sample was reported. The relative amount of the targetgene in each cell line and/or experiment was calibrated to controls. Alloligonucleotide primers were synthesized by Integrated DNA Technologies(Coralville, Iowa). GAPDH (Vandesompele et al., Genome Biol 3,RESEARCH0034 (2002)), PSA (Specht et al., Am J Pathol 158, 419-29(2001)), ERG (Exon 5-6_f and Exon 5-6_r) and ETV1 (Exon 6-7_f and Exon6-7_r) primers (Tomlins et al., Science 310, 644-8 (2005)) were asdescribed.

Fluorescence In Situ Hybridization (FISH)

Metaphase spreads were prepared from normal peripheral lymphocytes(NPLs) and LNCaP cells using standard techniques. Slides were treatedwith 2×SSC for 2 min, 70% ethanol for 2 min and 100% ethanol for 2 minbefore addition of the probe. Slides were coverslipped and incubated at750 for 2 min and hybridized overnight at 37° C. Post-hybridizationwashing was with 2×SSC at 42° C. for 5 min, followed by 3 washes inPBST. Fluorescent detection was performed using anti-digoxigeninconjugated to fluorescein (Roche Applied Science, Indianapolis, Ind.)and streptavidin conjugated to Alexa Fluor 594 (Invitrogen, Carlsbad,Calif.). Slides were counterstained and mounted in ProLong Gold AntifadeReagent with DAPI (Invitrogen). Slides were examined using a Zeiss AxioImager Z1 fluorescence microscope (Zeiss, Thornwood, N.Y.) and imagedwith a CCD camera using ISIS software (Metasystems, Altlussheim,Germany).

All BACs were obtained from the BACPAC Resource Center (Oakland, Calif.)and probe locations were verified by hybridization to metaphase spreadsof normal peripheral lymphocytes. For hybridization to the ETV1 regionon chromosome 7p, four BACs were used (telomeric to centromeric):RP11-124L22, RP11-313C20, RP11-703A4 and RP11-1149J13. For localizationto chromosome 14q, the FISH mapped BAC RP11-483K13, which we alsoconfirmed as hybridizing to 14q using NPLs. BAC DNA was isolated using aQIAFilter Maxi Prep kit (Qiagen, Valencia, Calif.) and probes weresynthesized using digoxigenin- or biotin-nick translation mixes (RocheApplied Science).

B. Results

Results are shown in FIGS. 26-28. FIG. 26 shows the over-expression andandrogen regulation of ETV1 in the LNCaP prostate cancer cell line. FIG.26A shows expression signature of androgen-regulated genes in VCaP andLNCaP prostate cancer cell lines. Heatmap of genes showing induction orrepression in either cell line (3,499 features, p<0.05 and fold changeratio>=1.5) by 1 nM synthetic androgen R1881 (green) compared to vehicletreatment (gray). Each row represents a gene; each column represents asample. Yellow and blue cells indicate over- or under-expression,respectively, according to the color scale. Gray cells indicate missingdata. Values for each cell line are centered on the correspondingcontrol samples. The locations of PSA, ERG and ETV1 in the heatmap areindicated and their expression is shown in the inset. FIG. 26B showsconfirmation of PSA induction by androgen in both VCaP and LNCaP cellsby quantitative PCR (QPCR). The relative expression of PSA (normalizedto GAPDH) in LNCaP (red) and VCaP (blue) cell lines was determined byQPCR. Cells were treated with vehicle or 1 nM R1881 for 48 hours in thepresence or absence of the anti-androgens Casodex or Flutamide asindicated. The relative amount of PSA in each sample was calibrated tothe amount in the control sample for each cell line. FIG. 26C shows ETV1induction by androgen in LNCaP cells. Using the same samples as B, therelative amount of ETV1 was determined by QPCR. FIG. 26D shows that ETV1is markedly over-expressed in LNCaP cells. The relative expression ofPSA, ETV1 and ERG were determined in the 48 hour control samples fromeach cell line by QPCR. The relative amount of target gene in eachsample was calibrated to the average amount of PSA from both cell lines.The fold difference in ERG and ETV1 expression between LNCaP and VCaP isindicated.

FIG. 27 shows rearrangement of ETV1 in LNCaP cells. FIG. 27A shows aschematic of BACs used as probes for fluorescence in situ hybridization(FISH). The location and coordinates at 7p21 (including the ETV1 locusand surrounding BACs) and 14q32 was determined on the May 2004 freeze ofthe human genome using the UCSC Genome Browser. BACs used in this studyare indicated as numbered rectangles. The location of ETV1 and DGKB areshown with the arrowhead indicating the direction of transcription. FIG.27B shows that RP11-124L22 and RP11-1149J13 co-localize to chromosome 7in normal peripheral lymphocytes (NPLs). Localization of RP11-124L22(BAC #1) and RP11-1149J13 (BAC #4) on metaphase spreads (top panel) orinterphase cells (bottom panel) was determined by FISH in NPLs. For allmetaphase pictures, signals on chromosome 7 are indicated by arrows,while signals on chromosome 14 are indicated by arrowheads of thecorresponding probe color. Higher magnification of informative regionsof metaphase spreads are shown in boxes. FIG. 27C shows localization ofBAC #1 and BAC #4 on metaphase spreads (top panel) and interphase cells(bottom panel) was determined in the near tetraploid LNCaP cell line.Two co-localized signals on chromosome 7, two red signals on chromosome7 and two green signals on a different chromosome were observed. FIG.27D shows signal from RP11-124L22 localizes to chromosome 14 in LNCaPcells. As in C, except RP11-124L22 (BAC #1) was co-hybridized withRP11-483K13 (BAC #5, FISH mapped to chromosome 14q) on LNCaP metaphasespreads. Four red signals from RP11-483K13 localize to chromosome 14q;two green signals localize to chromosome 7p and two green signalslocalize to chromosome 14q.

FIG. 28 shoes that the entire ETV1 locus is inserted into chromosome 14in LNCaP cells. FIG. 28A shows a schematic of BACs used in thisexperiment. FIG. 28B shows localization of RP11-124L22 (BAC #1) andRP11-313C20 (BAC #2) on metaphase spreads (top panel) and interphasecells (bottom panel) was determined by FISH in LNCaP cells. In metaphasespreads, two pairs of co-localized signals were observed on chromosome 7(yellow arrows) and chromosome 14 (yellow arrowheads).

These results demonstrate that the entire ETV1 locus is translocatedfrom chromosome 7 to chromosome 14. Although the genomic sequenceupstream of the insertion on chromosome 14 is unknown, it is likely thatthis region contains AREs, which drive the high level of ETV1 observedonly in LNCaP cells and the androgen responsiveness. These resultssuggest that LNCaP cells find use as an in vitro model of ETS genefusions seen in human prostate cancers.

Example 15 Knockdown of ETS Family Members in Prostate Cancer

This Example describes the knockdown of ETS family members in prostatecancer. siRNAs were used to knockdown expression of ETV1 and ERG inLnCaP and VCAP. Quantitative PCR was used to confirm the knockdown.Results are shown in FIGS. 29 and 30. The knockdown did not affectproliferation. Lentivirus expressing shRNA are generated for stableknockdowns.

Microarrays were performed on Agilent 44K Whole Genome arrays todetermine which genes were differentially expressed when ERG expressionwas knocked down in VCaP cells (which have the TMPRSS2:ERG fusion). Forthis experiment, three conditions were used: knockdown using DharmaconsiRNA for ERG (ERGsi), knockdown of luciferase (control), anduntransfected (untrans) VCaP cells. Three hybridizations of ERG/untransand two of control/untrans were performed. The genes were called aspresent in all five experiments, had standard deviations less than 0.5(of the average for both conditions), and showed a fold differencebetween the ERG and control of <0.75 or >1.5. The ERGdif field indicatesthe fold difference between the ERG and control knockdown experiments,so value less than one means the gene is underexpressed in the ERGknockdown (ERG itself ranks 81st in this analysis).

Example 16 Transgenic Mice

Transgenic mice that over express gene fusions of the present invention,as well as ETS and androgen responsive genes are generated. FIG. 31shows viral overexpression systems for use in generating mice. FIG. 32shows a schematic of genomic insertions in transgenic mice. Such micefind use in research (e.g., mechanistic studies) and drug screeningapplications.

Example 17 Identification of TMPRSS2:ERGa

As described above (Example 1), fusions of TMPRSS2 to ERG were observed.To determine the expressed protein from the TMPRSS2:ERGa gene fusion,PCR was used to amplify the portion of ERG (NM_(—)004449) from thefusion breakpoint at the beginning of exon 4 to the presumed stop codonin exon 11, inserting a 3× Flag tag immediately upstream of the stopcodon, from the VCaP prostate cancer cell line. The product was TAcloned into pCR8/GW/TOPO TA (Invitrogen) and bi directionally sequenced.Sequencing revealed the presence of two distinct isoforms, hereindesignated as ERG1 (includes exon 6 from ERG isoform 1 (NM_(—)182918,GGGGTGCAGCTTTTATTTTCCCAAATACTTCAGTATATCCTGAAGCTACGCAAAGAATTACAACTAGGCCAG; SEQ ID NO:73) and ERG2 (does not include this exon). Theproduct was Gateway cloned into the pLenti6/V5-DEST destination vector.This plasmid was transfected directly into PHINX cells for ERG proteinproduction.

A. Methods

Transfection Assay Phinx cells were transfected with either ERG2 or theempty vector using Fugene transfection reagent (Roche) as permanufacturer's instructions. A total of ten 150 mm diameter plates wereused for each construct. The cells were harvested 48 h post-transfectionand used for immunoprecipitation assay as described below.

Protein Lysis and Immunoprecipitation: Cells were washed in ice cold PBScontaining protease inhibitors and lysed by homogenization in TBScontaining 1% NP40. The supernantant containing proteins were estimatedfor their protein content using Bradfords Protein Assay (BioradLaboratories, Hercules, Calif.) as per manufacturer's instructions.Equal amounts of protein (approximately 30 mg in 15 ml buffer) from allsamples were used for immunoprecipitation studies. About 200 microlitresof a 50% slurry of EZVIEW Red ANTI-FLAG M2 Affinity Gel (Sigma, StLouis, Mo.) was added to each sample and incubated overnight at 4 C. Theimmunoprecipitate was washed thrice each with TBS containing 0.1% NP40and TBS alone. Bound proteins were eluted using FLAG peptide (Sigma, StLouis, Mo.) as per manufacturer's instruction. The elution was performedthree times. Proteins in the eluate were precipitated using 50% TCA(Sigma, St Louis, Mo.). The precipitate was washed thrice with ice coldacetone, resuspended in Laemmeli buffer and electrophoresed on 4-20%BIS-TRIS gel (Invitrogen Corporation, Carlsbad, Calif.). The gels werestained with mass spectrometry compatible silver stain (Silver Quest,Invitrogen Corporation, Carlsbad, Calif.). Bands corresponding to ERG2and the corresponding region in the vector lane were excised into 6pieces of 1 cm each. Each of the gel pieces were labeled bands 1-6starting from higher molecular weight region on the gel moving down.Thus Band 1 corresponds to the region containing high molecular weightproteins while band 6 corresponds to region of low molecular weight.Based on its native molecular mass of ERG2 (approximately 55 KDa) wouldmigrate in Bands 4 and 5. ERG2 sequence identification was repeatedthree times and the data was consolidated from all the experiments.

Protein Identification

The gel bands were collected, destained using the destaining solutionprovided in the Silver Stain Kit as per manufacturers instruction(Invitrogen Corporation, Carlsbad, Calif.). In gel digestion wasperformed using Porcine Trypsin (1:50, Promega Corporation, Madison,Wis.) in IM Ammonium Bicarbonate, pH 9. The digestion was performed for16 h at 37° C. At the end of 24 h the trypsin activity was stopped using3% formic acid. The peptides were extracted using 50% Acetonitrile. Thepeptides were dried and resuspended in 2% Acetonitrile containing 0.1%formic acid and separated by reversed-phase chromatography using a 0.075mm×150 mm C18 column attached to a Paradigm HPLC pump (Michrome BioResources Inc.). Peptides were eluted using a 45-min gradient from 5 to95% B (0.1% formic acid/95% acetonitrile), where solvent A was 0.1%formic acid/2% acetonitrile. A Finnigan LTQ mass spectrometer (ThermoElectron Corp.) was used to acquire spectra, the instrument operating indata-dependent mode with dynamic exclusion enabled. The MS/MS spectra onthree most abundant peptide ions in full MS scan were obtained. Thespectra are searched using the MASCOT search tool against the composite,non-identical NCBI human reference sequence database. These databasesearch results are validated for peptide assignment accuracy using thePeptideProphet program. This is a mixture model; an expectationmaximization evaluation assigning a probability of correct peptideidentification based on search result scores and various peptidefeatures including the number of typtic termini. A second program,ProteinProphet, is used to group peptides by protein and combine theirprobabilities to assign a probability of a correct protein assignment.Discriminatory power increases with the subsequent re-estimation ofindividual peptide probabilities by way of their NSP value, or number ofsibling peptides, which amounts to peptide grouping information and thestatus of a possible multi-hit protein.

Results:

TABLE 14 COVERAGE MAP (ERG2) SEQ NCBI N-terminal, ID MIQTVPDPAA HI . . .(SEQ ID NO:74) BAND05-20060217 NOMASTIKEALS VVSEDQSLFE CAYGTPHLAK TEMTASSSSD  40  74                                      SSSSD BAND03-20060206  75YGQTSKMSPR VPQQDWLSQP PARVTIKMEC NPSQVNGSRN  80  76           VPQQDWLSQP PAR BAND01-20060217  77            VPQQDWLSQP PARBAND02-20060206  78            VPQQDWLSQP PAR BAND02-20060209  79           VPQQDWLSQP PAR BAND02-20060217  80 YGQTSKMS   VPQQDWLSQP PARBAND03-20060206  81            VPQQDWLSQP PAR BAND03-20060209  82           VPQQDWLSQP PAR BAND03-20060217  83            VPQQDWLSQP PARBAND04-20060206  84            VPQQDWLSQP PAR    MEC NPSQVNGSRBAND04-20060209  85            VPQQDWLSQP PAR BAND04-20060217  86           VPQQDWLSQP PAR BAND05-20060217  87      SPDECSVAKG GKMVGSPDTV GMNYGSYMEE KHMPPPNMTT 120  88                                  HMPPPNMTT BAND01-20060206  89                                  HMPPPNMTT BAND02-20060206  90                                  HMPPPNMTT BAND02-20060209  91                        NYGSYMEE KHMP BAND02-20060217  92             MVGSPDTV GMNYGSYMEE KHMPPPNMTT BAND03-20060206  93                                  HMPPPNMTT BAND03-20060209  94                                  HMPPPNMTT BAND04-20060206  95             MVGSPDTV GMNYGSYMEE KHMPPPNMTT BAND04-20060209  96             MVGSPDTV GMNYGSYMEE KHMPPPNMTT BAND04-20060217  97NERRVIVPAD PTLWSTDHVR QWLEWAVKEY GLPDVNILLF 160  98 NER VIVPADPTLWSTDHVR QWLEWAVKEY GLPDVNILLF BAND01-20060206  99NER                           EY GLPDVNILLF BAND02-20060206 100 NERBAND02-20060209 101 NER VIVPAD PTLWSTDHVR QWLEWAVK BAND03-20060206 102NERRVIVPAD PTLWSTDHVR         EY GLPDVNILLF BAND03-20060209 103 NERVIVPAD PTLWSTDHVR QWLEWAVKEY GLPDVNILLF BAND04-20060206 104 NERRVIVPADPTLWSTDHVR QWLEWAVKEY GLPDVNILLF BAND04-20060209 105 NERRVIVPADPTLWSTDHVR BAND04-20060217 106                               EYGLPDVNILLF BAND05-20060206 107QNIDGKELCK MTKDDFQRLT PSYNADILLS HLHYLRETPL 200 108QNIDGK             LT PSYNADILLS HLHYLRETPL BAND01-20060206 109                                       ETPL BAND01-20060217 110QNIDGK                                 ETPL BAND02-20060206 111                                       ETPL BAND02-20060217 112                                       ETPL BAND03-20060206 113QNIDGK             LT PSYNADILLS HLHYLRETPL BAND03-20060209 114                                       ETPL BAND03-20060217 115QNIDGK             LT PSYNADILLS HLHYLRETPL BAND04-20060206 116QNIDGK             LT PSYNADILLS HLHYLRETPL BAND04-20060209 117                   LT PSYNADILLS HLHYLRETPL BAND04-20060217 118 QNIDGKBAND05-20060206 119                       PSYNADILLS HLHYLRETPLBAND05-20060217 120 PHLTSDDVDK ALQNSPRLMH ARNTGGAAFI FPNTSVYPEA 240 121                PRLMH ARNT BAND01-20060206 122 PHLTSDDVDKBAND01-20060206 123 PHLTSDDVDK ALQNSPR BAND01-20060217 124 PHLTSDDVDKALQNSPR BAND02-20060206 125 PHLTSDDVDK ALQNSPR BAND02-20060217 126PHLTSDDVDK ALQNSPR BAND03-20060206 127 PHLTSDDVDK BAND03-20060209 128PHLTSDDVDK ALQNSPR BAND03-20060217 129 PHLTSDDVDK ALQNSPRBAND04-20060206 130 PHLTSDDVDK ALQNSPR BAND04-20060209 131                       RNT BAND0420060209 132 PHLTSDDVDK ALQNSPRBAND04-20060217 133 PHLTSDDVDK ALQNSPRL BAND05-20060217 134TQRITTRPDL PYEPPRRSAW TGHGHPTPQS KAAQPSPSTV 280 135         DLPYEPPRBAND01-20060206 136                   SAW TGHGHPTPQS KAAQPSPSTVBAND01-20060206 137            PYEPPRR BAND01-20060217 138                  SAW TGHGHPTPQS KAAQPSPSTV BAND02-20060206 139                  SAW TGHGHPTPQS KAAQPSPSTV BAND02-20060209 140           PYEPPRRSAW TGHGHPTPQS KAAQPSPSTV BAND02-20060217 141                  SAW TGHGHPTPQS KAAQPSPSTV BAND03-20060206 142                  SAW TGHGHPTPQS KAAQPSPSTV BAND03-20060209 143        DL PYEPPRR BAND03-20060217 144            PYEPPRRSAW TGHGHPTPQSKAAQPSPSTV BAND04-20060206 145         DL PYEPPRRSAW TGHGHPTPQSKAAQPSPSTV BAND04-20060209 146                  DL PYEPPRRBAND04-20060209 147         DL PYEPPRRSAW TGHGHPTPQS KAAQPSPSTVBAND04-20060217 148                                   AAQPSPSTVBAND05-20060206 149                   SAW TGHGHPTPQS KAAQPSPSTVBAND05-20060209 150         DL PYEPPRRSAW TGHGHPTPQS KAAQPSPSTVBAND05-20060217 151                   SAW TGHGHPTPQS KAAQPSPSTVBAND06-20060209 PKTEDQRPQL DPYQILGPTS SRLANPGSGQ IQLWQFLLEL 320 152 PKBAND01-20060206 153   TEDQRPQL DPYQILGPTS SR BAND01-20060217 154PKTEDQRPQL DPYQILGPTS SR BAND02-20060206 155 PKTEDQRPQL DPYQILGPTS SRBAND02-20060209 156 PKTEDQRPQL DPYQILGPTS SR BAND02-20060217 157PKTEDQRPQL DPYQILGPTS SR BAND03-20060206 158 PKTEDQRPQL DPYQILGPTS SRBAND03-20060209 159   TEDQRPQL DPYQILGPTS SR BAND03-20060217 160PKTEDQRPQL DPYQILGPTS SR BAND04-20060206 161 PKTEDQRPQL DPYQILGPTS SRBAND04-20060209 162 PKTEDQRPQL DPYQILGPTS SR BAND04-20060217 163 PKBAND05-20060206 164 PKTEDQRPQL DPYQILGPTS SR BAND05-20060209 165PKTEDQRPQL DPYQILGPTS SR BAND05-20060217 166 PK BAND06-20060209 167LSDSSNSSCI TWEGTNGEFK MTDPDEVARR WGERKSKPNM 360 168                      MTDPDEVAR BAND01-20060206 169                      MTDPDEVAR BAND02-20060206 170                      MTDPDEVAR BAND03-20060206 171                      MTDPDEVAR BAND03-20060209 172                      MTDPDEVAR BAND04-20060206 173                      MTDPDEVARR BAND04-20060209 174                       TDPDEVARR     KSKPNM BAND04-20060217 175                      MTDPDEVAR BAND05-20060209 176                                     KSKPNM BAND05-20060217 177NYDKLSRALR YYYDKNIMTK VHGKRYAYKF DFHGIAQALQ 400 178                               F DFHGIAQALQ BAND02-20060206 179                               F DFHGIAQALQ BAND02-20060209 180                               F DFHGIAQALQ BAND03-20060206 181                               F DFHGIAQALQ BAND03-20060209 182           YYYDKNIMTK      YAYKF DFHGIAQALQ BAND04-20060209 183 NYDKLSRBAND04-20060217 184 NYDKLSR    YYYDKNIMTK BAND05-20060217 185PHPPESSLYK YPSDLPYMGS YHAHPQKMNF VAPHPPALPV 440 186 PHPPESSLYKBAND02-20060206 187 PHPPESSLYK YPSDLPYMGS YHAH BAND02-20060209 188PHPPESSLYK YPSDLPYMGS YHAHPQK BAND03-20060206 189 PHPPESSLYK YPSDLPYMGSYHAHPQK BAND03-20060209 190            YPSDLPYMGS YHAHPQKBAND04-20060206 191 PHPPESSLYK YPSDLPYMGS YHAHPQK BAND04-20060209 192TSSSFFAAPN PYWNSPTGGI YPNTRLPTSH MPSHLGTYY 479 193               NSPTGBAND02-20060217 194                SPTGGI YPNTR BAND04-20060209 195NOTE: E*BAND*-* represent ERG2 peptides in ERG1 experimentsThe table shows the coverage map for ERG2 obtained over 3 differentexperiments. The underlined amino acid sequence corresponds to the insilico translated sequence of ERG1 that was cloned from VCAP cells. Theamino acid sequence GGAAFIFPNTSVYPEATQRITTRP (SEQ ID NO: 196)corresponds to the exon that is specific to ERG1 and is missing in ERG2.The remaining amino acid sequence correspond to ERG2 sequence identifiedin each of the three experiments. ERG2 was identified in Bands 1-5 inall the experiments. The peptide sequences for ERG2 obtained in each ofthese bands is illustrated. A very high coverage of the ERG2 protein wasobserved over the three experiments. The coverage map showed that thecoverage of peptides in the N-terminal region of the cloned protein,corresponding to the first 50 amino acid residues were rarely observedin the mass spectrometry coverage map. However, the peptide VPQQDWLSQP(SEQ ID NO: 197) that starts with amino acid valine was found to behighly abundant and thus identified in all the experiments. Closerevaluation suggested that amino acid in the 47^(th) position was an inframe Methionine. The lack of any peptide upstream (Nterminus) of the 47th methionine in multiple experiments confirms that it is the N-terminalamino acid of ERG2. Further, the presence of a Arginine residue at the50^(th) position makes it a potential tryptic cleavage site. Digestionby trypsin at this site would result in a shorter N-terminal peptideMSPR, which is too small for identification by ion trap massspectrometer and a longer C-terminal peptide VPQQDWLSQP (SEQ ID NO:198), which was identified in all the experiments. Also the peptidesequence MIQTVPDPAA HI (SEQ ID NO: 199) was identified in a singleexperiment at a very low probability score. This maps to the N-terminusof ERG as reported in NCBI. This sequence was not a part of theectopically overexpressed construct that was cloned from the VCAP cells.This could have been obtained from the in vivo ERG that is expressed inPHINX cells and thus may represent part of the ERG associated withbenign cells.

Thus, in summary, the results indicate that the third Methionine is thetranslational Start site for the TMPRSS2-ERG fusion product. MASTIKEALSVVSEDQSLFE CAYGTPHLAK TEMTA YGQTSKMSPR VPQQDWLSQP (SEQ ID NO:200). TheFirst Methionine is the translational START Site for endogenous ERG.MIQTVPDPAA HI (SEQ ID NO:201). FIG. 20 shows a schematic of theendogenous and fusion polypeptides.

Example 18 FISH Analysis on Urine Samples

To isolate and prepare prostatic cells from urine, ˜30 ml of urine iscollected following an attentive digital rectal exam. Immediately, 15 mlof PreservCyt is added, and the sample is centrifuged at 4000 rpm in a50 ml tube for 10 min at room temperature. The supernatant is discarded,the pellet is resuspended in 15 ml of 0.75 M KCl for 15 min at roomtemperature, and centrifuged at 4000 rpm in a 50 ml tube for 10 min atroom temperature. The supernatant is discarded, and the pellet isresuspended in 10 ml of a 3:1 ratio of methanol:glacial acetic acid.This is then centrifuged at 4000 rpm for 8 min. The supernatant isdiscarded, except for 200 μl, and the pellet is resuspended. Theresuspended pellet is then dropped onto glass slides and allowed to airdry. Hybridization and probe preparation are as in Example 2 above, withthe ERG 5′/3′ and TMPRSS 5′/3′ probe pairs.

Example 19 Additional ETV1 Gene Fusions A. Materials and Methods Samplesand Cell Lines

Prostate tissues were from the radical prostatectomy series at theUniversity of Michigan and from the Rapid Autopsy Program1, which areboth part of University of Michigan Prostate Cancer Specialized Programof Research Excellence (S.P.O.R.E.) Tissue Core. All samples werecollected with informed consent of the patients and prior institutionalreview board approval.

The benign immortalized prostate cell line RWPE and the prostate cancercell lines LNCaP, Du145 NCI-H660 and PC3 were obtained from the ATCC.Primary benign prostatic epithelial cells (PrEC) were obtained fromCambrex Bio Science (Walkersville, Md.). The prostate cancer cell linesC4-2B, LAPC4 and MDA-PCa 2B were provided by Evan Keller (University ofMichigan). The prostate cancer cell line 22-RV1 was provided by JillMcKoska (University of Michigan). VCaP was derived from a vertebralmetastasis from a patient with hormone-refractory metastatic prostatecancer (Korenchuk et al., In Vivo 15, 163-8 (2001)).

For androgen stimulation experiments, LNCaP cells were grown incharcoal-stripped serum containing media for 24 hours before treatmentfor 24 hours with 1% ethanol or 1 nM of methyltrienolone (R1881, NENLife Science Products, Boston, Mass.) dissolved in ethanol. For allsamples, total RNA was isolated with Trizol (Invitrogen, Carlsbad,Calif.) according to the manufacturer's instructions.

Quantitative PCR (QPCR)

Quantitative PCR (QPCR) was performed using Power SYBR Green Mastermix(Applied Biosystems, Foster City, Calif.) on an Applied Biosystems 7300Real Time PCR system as described (Tomlins et al., Cancer Res 66,3396-400 (2006); Tomlins et al., Recurrent fusion of TMPRSS2 and ETStranscription factor genes in prostate cancer. Science 310, 644-8(2005)). All oligonucleotide primers were synthesized by Integrated DNATechnologies (Coralville, Iowa) and are listed in Table 15. HMBS andGAPDH5, and PSA6 primers were as described. Androgen stimulationreactions were performed in quadruplicate, all other reactions wereperformed in duplicate.

RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE)

RLM-RACE was performed using the GeneRacer RLM-RACE kit (Invitrogen),according to the manufacturer's instructions as described (Tomlins etal., 2005, supra; Tomlins et al., 2006, supra). To obtain the 5′ end ofETV1, first-strand cDNA was amplified with Platinum Taq High Fidelity(Invitrogen) using the GeneRacer 5′ primer and ETV1_exon4-5_r. Productswere cloned and sequenced bidirectionally as described (Tomlins et al.,2005, supra; Tomlins et al., 2006, supra). RLM-RACEd cDNA was not usedfor other assays.

Fluorescence In Situ Hybridization (FISH)

Interphase FISH on formalin-fixed paraffin-embedded (FFPE) tissuesections was performed as described ((Tomlins et al., 2005, supra). Aminimum of 50 nuclei per assay were evaluated. Metaphase spreads ofLNCaP and MDA-PCa 2B were prepared using standard cytogenetictechniques. Slides were pre-treated in 2×SSC for 2 min, 70% ethanol for2 min and 100% ethanol for 2 min, and air dried. Slides and probes wereco-denatured at 75 degrees for 2 min, and hybridized overnight at 37° C.Post-hybridization was in 0.5×SSC at 42° C. for 5 min, followed by 3washes in PBST. Fluorescent detection was performed usinganti-digoxigenin conjugated to fluorescein (Roche Applied Science,Indianapolis, Ind.) and streptavidin conjugated to Alexa Fluor 594(Invitrogen). Slides were counterstained and mounted in ProLong GoldAntifade Reagent with DAPI (Invitrogen). Slides were examined using aZeiss Axio Imager Z1 fluorescence microscope (Zeiss, Thornwood, N.Y.)and imaged with a CCD camera using ISIS software (Metasystems,Altlussheim, Germany). BACs (listed in Table 16) were obtained from theBACPAC Resource Center (Oakland, Calif.), and probes were prepared asdescribed (Tomlins et al., 2005, supra). Pre-labeled chromosome 7centromere and 7p telomeric probes were obtained from Vysis (DesPlaines, Ill.). The integrity and correct localization of all probeswere verified by hybridization to metaphase spreads of normal peripherallymphocytes.

Tissue Specific Expression

To determine the tissue specific expression of 5′ fusion partners andgenes at 14q13-q21, the International Genomics Consortium's expOdataset, consisting of expression profiles from 630 tumors of 29distinct types, using the Oncomine database. To interrogate theexpression of HERV-K_(—)22q11.23, which is not monitored by commercialarray platforms, the Lynx Therapeutics normal tissue massively parallelsignature sequencing (MPSS) dataset (GSE1747) was queried with the MPSStag “GATCTTTGTGACCTACT” (SEQ ID NO:308), which unambiguously identifiesHERV-K_(—)22q11.23, as described (Stauffer et al., Cancer Immun 4, 2(2004)). Descriptions of tumor types from the expO dataset and thenormal tissue types from the MPSS dataset are provided in Table 17.

Expression Profiling

Expression profiling of LNCaP, C4-2B, RWPE-ETV1 and RWPE-GUS cells wereperformed using the Agilent Whole Human Genome Oligo Microarray (SantaClara, Calif.). Total RNA isolated using Trizol was purified using theQiagen RNAeasy Micro kit (Valencia, Calif.). One μg of total RNA wasconverted to cRNA and labeled according to the manufacturer's protocol(Agilent). Hybridizations were performed for 16 hrs at 65° C., andarrays were scanned on an Agilent DNA microarray scanner. Images wereanalyzed and data extracted using Agilent Feature Extraction Software9.1.3.1, with linear and lowess normalization performed for each array.For the LNCaP and C4-2B hybridizations, the reference for each cell linewas pooled benign prostate total RNA (Clontech, Mountain View, Calif.).A dye flip for each cell line was also performed. Features were rankedby average expression (Log Ratio) in the two LNCaP arrays divided by theaverage expression in the two C4-2B arrays after correction for the dyeflip. For RWPE cells, four hybridizations were performed (duplicateRWPE-ETV1/RWPE-GUS and RWPE-GUS/RWPE-ETV1 hybridizations). Over andunder-expressed signatures were generated by filtering to include onlyfeatures with significant differential expression (PValueLogRatio<0.01)in all four hybridizations and 2 fold average over- or under-expression(Log Ratio) after correction for the dye flip.

Southern Hybridization

Genomic DNA (10 μg) from LNCaP, VCaP, pooled normal human male DNA(Promega, Madison, Wis.) and normal placental DNA (Promega) was digestedwith EcoRI or PstI (New England Biologicals, Ipswich, Mass.) overnight.Fragments were resolved on a 0.8% agarose gel at 40 V overnight,transferred to Hybond NX nylon membrane, prehybridized, hybridized withprobe and washed according to standard protocols. A series of 22 probesspanning the region of chr 7 implicated by FISH (between RP11-313C20 andRP11-703A4) were generated by PCR amplification with Platinum Taq HighFidelity on pooled normal human male genomic DNA (Table 15). Twenty-fiveng of each probe was labeled with dCTP-P32 and used for hybridization.

Inverse PCR

To identify the ETV1 breakpoint in LNCaP cells an inverse PCR strategybased on the rearrangement identified by Southern blotting was used.Primers A1, A2, A3, which are reverse complemented from the wildtypesequence and are divergent to primers B1, B2, B3, were used for inversePCR on PstI digested and religated (in order to promoter intramolecularligation) LNCaP genomic DNA template. Nested PCRs were performed in thefollowing order of primer combinations: A1-B1, A2-B2 and A3-B3. Expand20 kbplus PCR system (Roche Diagnostics GmbH, Mannheim, Germany) wasused for amplifying the fusion product according to the manufacturessuggestions. The enriched 3 Kb band observed in nested PCRs was clonedinto pCR8/GW/TOPO (Invitrogen), miniprep DNA was screened for insertsand positive clones were sequenced (University of Michigan DNASequencing Core, Ann Arbor, Mich.). Fusion was then confirmed by PCRwith Platinum Taq High Fidelity using fusion specific primers (Table15).

In Vitro Over-Expression of ETV1

cDNA of ETV1, as present in the TMPRSS2:ETV1 fusion to the reported stopcodon of ETV1 (269-1521, NM_(—)004956.3), was amplified by RT-PCR fromMET264 and TOPO cloned into the Gateway entry vector pCR8/GW/TOPO(Invitrogen), yielding pCR8-ETV1. To generate adenoviral and lentiviralconstructs, pCR8-ETV1 and a control entry clone (pENTR-GUS) wererecombined with pAD/CMV/V5 (Invitrogen) and pLenti6/CMV/V5 (Invitrogen),respectively, using LR Clonase II (Invitrogen). Control pAD/CMV/LACZclones were obtained from Invitrogen. Adenoviruses and Lentiviruses weregenerated by the University of Michigan Vector Core. The benignimmortalized prostate cell line RWPE was infected with lentivirusesexpressing ETV1 or GUS, and stable clones were generated by selectionwith blasticidin (Invitrogen). Benign PrEC were infected withadenoviruses expressing ETV1 or LACZ, as stable lines could not begenerated in primary PrEC cells. Cell counts were estimated bytrypsinizing cells and analysis by Coulter counter at the indicated timepoints in triplicate. For invasion assays, equal numbers of PREC-ETV1and -LACZ (48 hours after infection) or stable RPWE-ETV1 and -GUS cellswere seeded onto the basement membrane matrix (EC matrix, Chemicon,Temecula, Calif.) present in the insert of a 24 well culture plate, withfetal bovine serum added to the lower chamber as a chemoattractant.After 48 hours, non-invading cells and EC matrix were removed by acotton swab. Invaded cells were stained with crystal violet andphotographed. The inserts were treated with 10% acetic acid andabsorbance was measured at 560 nm.

ETV1 Knockdown

For siRNA knockdown of ETV1 in LNCaP cells, the individual siRNAscomposing the Dharmacon SMARTpool against ETV1 (MU-003801-01, Chicago,Ill.) were tested for ETV1 knockdown by qPCR, and the most effectivesingle siRNA (D-003801-05) was used for further experiments. siCONTROLNon-Targeting siRNA # 1 (D-001210-01) or siRNA against ETV1 wastransfected into LNCaP cells using Oligofectamine (Invitrogen). After 24hours a second identical transfection was carried out and cells wereharvested 24 hours later for RNA isolation and invasion assays asdescribed below. For shRNA knockdown of ETV1 in LNCaP cells, theshRNAmir construct against ETV1 from the pMS2 retroviral vector(V2HS_(—)61929, Open Biosystems, Huntsville, Ala.) was cloned into anempty pGIPZ lentivral vector (RHS4349, Open Biosystems) according to themanufacturer's protocol. pGIPZ lentiviruses with shRNAmirs against ETV1or a non silencing control (RHS4346) were generated by the University ofMichigan Vector Core. LNCaP cells were infected with lentiviruses, and48 hours later cells were used for invasion assays as described below.Representative results from 6 independent experiments are shown.

Invasion Assays

Equal numbers of the indicated cells were seeded onto the basementmembrane matrix (EC matrix, Chemicon, Temecula, Calif.) present in theinsert of a 24 well culture plate, with fetal bovine serum added to thelower chamber as a chemoattractant. After 48 hours, non-invading cellsand EC matrix were removed by a cotton swab. Invaded cells were stainedwith crystal violet and photographed. The inserts were treated with 10%acetic acid and absorbance was measured at 560 nm.

FACS Cell Cycle Analysis

RWPE-ETV1 and RWPE-GUS cells were assessed by FACS for cell cyclecharacterization. Cells were washed with 2×PBS and approximately 2×10⁶cells were resuspended in PBS before fixation in 70% ethanol. Pelletedcells were washed and treated with RNase (100 μg/ml final concentration)and propidium iodide (10 μg/ml final concentration) at 37° C. for 30min. Stained cells were analyzed on a LSR II flow cytometer (BDBiosciences, San Jose, Calif.) running FACSDivia, and cell cycle phaseswere calculated using ModFit LT (Verity Software House, Topsham, Me.).

Soft Agar Assay

A 0.6% (wt/vol) bottom layer of low melting point agarose in normalmedium was prepared in six-well culture plates. On top, a layer of 0.3%agarose containing 1×10⁴ RWPE-GUS, RWPE-ETV1 or DU145 (positive control)cells was placed. After 12 days, foci were stained with crystal violetand counted.

Immunoblot Analyses

Cells were homogenized in NP40 lysis buffer containing 50 mM Tris-HCl(pH 7.4), 1% NP40 (Sigma, St. Louis, Mo.), and complete proteinaseinhibitor mixture (Roche). Fifteen μg of protein extracts were mixedwith SDS sample buffer and electrophoresed onto a 10% SDS-polyacrylamidegel under reducing conditions. The separated proteins were transferredonto nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway,N.J.). The membrane was incubated for 1 h in blocking buffer[Tris-buffered saline with 0.1% Tween (TBS-T) and 5% nonfat dry milk].Primary antibody was applied at the indicated dilution in blockingbuffer overnight at 4° C. After washing three times with TBS-T buffer,the membrane was incubated with horseradish peroxidase-linked donkeyanti-mouse IgG antibody (Amersham Pharmacia Biotech) at a 1:5,000dilution for 1 hour at room temperature. The signals were visualizedwith the enhanced chemiluminescence detection system (Amersham PharmaciaBiotech) and autoradiography.

Mouse monoclonal anti-MMP-3 (IM36L, Calbiochem, San Diego) was appliedat 1:500 dilution and mouse monoclonal anti-uPA (IM13L, Calbiochem) wasapplied at 1:500 dilution, and mouse anti-GAPDH antibody (Abcam,Cambridge, Mass.) was applied at 1:30,000 dilution for loading control.

Transgenic ETV1 Mice

For in vivo over-expression of ETV1, a C terminal 3×FLAG-epitope taggedconstruct was generated by PCR using pCR8-ETV1 as the template with thereverse primer encoding a triple FLAG tag before the stop codon. Theproduct was TOPO cloned into pCR8. To generate a prostate specific ETV1transgenic construct, 3×FLAG-ETV1 was inserted into pBSII (Stratagene,La Jolla, Calif.) downstream of a modified small composite probasinpromoter (ARR2PB) and upstream of a bovine growth hormone polyA site(PA-BGH). The ARR2PB sequence contains the original probasin sequence PB(−426/+28) plus two additional androgen response elements. The constructwas sequenced and tested for promoter inducibility by androgen in LNCaPcells upon transient transfection before microinjection into FVB mouseeggs. The ARR2PB-ETV1 plasmid was linearized with PvuI/KpnI//SacII andmicroinjected into fertilized FVB mouse eggs and surgically transplantedinto a pseudo-pregnant female by the University of Michigan TransgenicAnimal Model Core. Transgenic founders were screened by PCR usinggenomic DNA isolated from tail snips. Transgenic ARR2PB-ETV1 founderswere crossed with FVB mice and the transgene-positive male miceoffspring were sacrificed at various time points. Prostates fromtransgenic mice were dissected using a Nikon dissection scope, fixed in10% buffered formalin and embedded in paraffin. Five μm sections werestained with hematoxylin and eosin, and evaluated by three pathologistsaccording to the criteria provided in The Consensus Report from the BarHarbor Meeting of the Mouse Models of Human Cancer Consortium ProstatePathology Committee (Nam et al., Cancer Biol Ther 6 (2007)).

For immunohistochemical detection of ETV1-FLAG, the basal cell markersp63 and cytokeratin 5 (CK5), and smooth muscle actin, deparaffinizedslides were subjected to microwave-citrate antigen retrieval andincubated with rabbit anti-FLAG polyclonal antibody (1:50 dilution,overnight incubation, Cell Signaling Technology, #2368), mousemonoclonal anti-p63 antibody (1:100 dilution, 45′ incubation, LabVision,MS1081P1), mouse monoclonal anti-smooth muscle actin antibody (1:50dilution, 30′ incubation, DakoAb M0851) and rabbit polyclonal anti-CK5antibody (1:500 dilution, 30′ incubation, AbCam, ab24647), respectively.Visualization of p63 and SMA was performed using a standardbiotin-avidin complex technique using M.O.M Immunodetection kit (PK2200,Vector Laboratories). FLAG and CK5 were detected usingEnvision+System-HRP (DAB) kit (K4011, DakoCytomation).

TABLE 15 SEQ Sequence ID Assay Gene/Region Sequence Bases Primer 5′ to3′ NO Androgen/ ETV1 NM_004956.3 624-645 ETV1_exon 6-7_f CTACCCCATGGACCA309 Expression CAGATTT QPCR Androgen/ ETV1 NM_004956.3 771-750 ETV1_exon6-7_r CTTAAAGCCTTGTGG 310 Expression TGGGAAG QPCR Expression ERGNM_004449.3 574-597 ERG_exon 5-6_f CGCAGAGTTATCGTG 311 QPCR CCAGCAGATExpression ERG NM_004449.3 659-636 ERG_exon 5-6_r CCATATTCTTTCACC 312QPCR GCCCACTCC RLM-RACE NA NA Generacer 5′_f CGACTGGAGCACGA 313GGACACTGA RLM-RACE ETV1 NM_004449.3 374-351 ETV1_exon 4-5_rCATGGACTGTGGGGT 314 TCTTTCTTG RLM-RACE ETV1 NM_004449.3 735-710ETV1_exon 7-r AGACATCTGGCGTTG 315 GTACATAGGAC Fusion QPCR HERV-BC020811.1 303-327 HERV-K:ETV1-f GAGTCCCAAGTACGT 316 K_22q11.23CCACGGTCAG Fusion QPCR ETV1 NM_004956.3 371-345 HERV-K:ETV1-rGGACTGTGGGGTTCT 317 TTCTTGATTTTC Fusion QPCR HNRPA2B1 NM_002137.2136-155 HNRPA2B1:ETV1-f TGCGGGAAATCGGG 318 CTGAAG Fusion QPCR ETV1NM_004956.3 181-154 HNRPA2B1:ETV1-r TTTTCCTGACATTTG 319 TTGGTTTCTCGTTFusion QPCR SLC45A3 NM_033102.2 74-92 SLC45A3:ETV1-f CGCTGGCTCCGGGTG 320ACAG Fusion QPCR ETV1 NM_004956.3 366-340 SLC45A3:ETV1-r GTGGGGTTCTTTCTT321 GATTTTCAGTGG Fusion QPCR C15ORF21 NM_001005266.1 313-336C15ORF21:ETV1-f CAACTAACACTGCG 322 GCTTCCTGAG Fusion QPCR ETV1NM_004956.3 483-461 C15ORF21:ETV1-r CATTCCCACTTGTGG 323 CTTCTGATAndrogen QPCR TMPRSS2 NM_005656.2 1539-1563 TMPRSS2-f CAGGAGTGTACGGG 324AATGTGATGGT Androgen QPCR TMPRSS2 NM_005656.2 1609-1585 TMPRSS2-rGATTAGCCGTGTGCC 325 CTCATTTGT Androgen QPCR TTC6 NM_001007795.11080-1108 TTC6-f TGCCATGAAGATCA 326 GTACTACAGCAGAAT Androgen QPCR TTC6NM_001007795.1 1150-1125 TTC6-r GTGGCCCATAAACTC 327 ATGAATCACC AndrogenQPCR SLC25A21 NM_030631.1 356-377 SLC25A21-f CAGATCGTGGCCGGT 328 GGTTCTAndrogen QPCR SLC25A21 NM_030631.1 408-483 SLC25A21-r GGGTGCATCAGGCA 329AATTTCTACAAG Androgen QPCR MIPOL1 NM_138731.2 1607-1633 MIPOL1-fCAACAACAAAATGA 330 GGAACTGGCTACT Androgen QPCR MIPOL1 NM_138731.21673-1649 MIPOL1-r ATTCCATATTTGCTC 331 GCTCTGTCAG Androgen QPCR FOXA1NM_004496.2 327-350 FOXA1-f GAAGATGGAAGGGC 332 ATGAAACCAG Androgen QPCRFOXA1 NM_004496.2 408-389 FOXA1-r GCTGACCGGGACGG 333 AGGAGT AndrogenQPCR SLC45A3 NM_033102.2 1223-1242 SLC45A3-f TCGTGGGCGAGGGG 334 CTGTAAndrogen QPCR SLC45A3 NM_033102.2 1308-1284 SLC45A3-r CATCCGAACGCCTTC335 ATCATAGTGT Androgen QPCR HERV- BC020811.1 168-194 HERV-CTTTTCTCTAGGGTG 336 K_22q11.23 K_22q11.23-f AAGGGACTCTCG Androgen QPCRHERV- BC020811.1 263-238 HERV- CTTCACCCACAAGGC 337 K_22q11.23K_22q11.23-r TCACTGTAGAC Androgen QPCR HNRPA2B1 NM_002137.2 594-620HNRPA2B1-f GCTTTGGCTTTGTTA 338 CTTTTGATGACC Androgen QPCR HNRPA2B1NM_002137.2 693-665 HNRPA2B1-r GCCTTTCTTACTTCT 339 GCATTATGACCATTAndrogen QPCR C15ORF21 NM_001005266.1 219-243 C15ORF21-f AAGGACGTGCAAGG340 ATGTTTTTATT Androgen QPCR C15ORF21 NM_001005266.1 293-274 C15ORF21-rATGGGAAGATGGGG 341 GCTGTT Southern probe Chr 7 (5′ to NT_007819.1613,685,335-13,685,364 LNCAP_A-f GTCAATGGCTAAAA 342 ETV1)GATGGATAAAAGTGGA Southern probe Chr 7 (5′ to NT_007819.1613,686,833-13,686,804 LNCAP_A-r CAGATAGAAGAGGG 343 ETV1)GTTAGCAAAATGTGTT Southern probe Chr 7 (5′ to NT_007819.1613,690,754-13,690,779 LNCAP_B1-f CAGAAGGCAAATGT 344 ETV1) GAGAGGATAGTCSouthern probe Chr 7 (5′ to NT_007819.16 13,691,679-13,691,657LNCAP_B1-r CTGGATCTGTAACAC 345 ETV1) CCGTGAGC Southern probe Chr 7(5′ to NT_007819.16 13,693,600-13,693,625 LNCAP_B2-f AAAAAGCAAAGACA 346ETV1) AGACCGTGGATT Southern probe Chr 7 (5′ to NT_007819.1613,695,054-13,695,028 LNCAP_B2-r GAACTACCTGCGTGC 347 ETV1) TGACTTGGAGATSouthern probe Chr 7 (5′ to NT_007819.16 13,699,722-13,699,801 LNCAP_C-fAAAAGGCAAAGAGG 348 ETV1) GGTTAAAACATACATA Southern probe Chr 7 (5′ toNT_007819.16 13,700,641-13,700,618 LNCAP_C-r AACCCCTCCTTCCAC 349 ETV1)TTCTCCACT Southern probe Chr 7 (5′ to NT_007819.16 13,705,005-13,705,034LNCAP_D-f TGGAGGCATAGAAA 350 ETV1) AGCTGAGAAATAAG Southern probe Chr 7(5′ to NT_007819.16 13,705,856-13,705,830 LNCAP_D-r TTGGTGCTAGAAGA 351ETV1) ACTGGGAGAAAC Southern probe Chr 7 (5′ to NT_007819.1613,711,263-13,711,291 LNCAP_F-f GAAAGTCAGGGGCA 352 ETV1) CATATAGATTAGAGSouthern probe Chr 7 (5′ to NT_007819.16 13,712,112-13,712,089 LNCAP_F-rGCCTTCCCCATACAG 353 ETV1) TTTCTCCTT Southern probe Chr 7 (5′ toNT_007819.16 13,708,247-13,708,275 LNCAP_E-f AAGTTCGTTAAGCCC 354 ETV1)AGGATCGTAGGTA Southern probe Chr 7 (5′ to NT_007819.1613,709,539-13,709,514 LNCAP_E-r ATATGAAGCCAGCA 355 ETV1) GCCAGGTAGCASouthern probe Chr 7 (5′ to NT_007819.16 13,719,560-13,719,587 LNCAP_G-fTTAGATAAACTGAA 356 ETV1) AGCCGAACCTGAAC Southern probe Chr 7 (5′ toNT_007819.16 13,720,465-13,720,441 LNCAP_G-r CAAACTGGCAAGCA 357 ETV1)ATGTGAACTGT Southern probe Chr 7 (5′ to NT_007819.1613,723,583-13,723,615 LNCAP_H-f TCACCGACAAAACC 358 ETV1) CATAGAGAAAGAGTSouthern probe Chr 7 (5′ to NT_007819.16 13,724,850-13,724,823 LNCAP_H-rTTAAATGGTGAGGC 359 ETV1) AATGAGGAAAGTG Southern probe Chr 7 (5′ toNT_007819.16 13,727,707-13,727,735 LNCAP_I-f TTGCTCATTCTCTTTC 360 ETV1)TCCCCTACACTAA Southern probe Chr 7 (5′ to NT_007819.1613,729,407-13,729,387 LNCAP_I-r TCCCCACCACCAACC 361 ETV1) ATCCTCSouthern probe Chr 7 (5′ to NT_007819.16 13,730,208-13,730,233LNCAP_JI-f CTGGGGGAAAAGCA 362 ETV1) AGTAGGAAAGTA Southern probe Chr 7(5′ to NT_007819.16 13,731,073-13,731,047 LNCAP_JI-r ACAAGAGTTAGTCA 363ETV1) CGGCAAAGGAGTT Southern probe Chr 7 (5′ to NT_007819.1613,733,199-13,733,220 LNCAP_J2-f GCCCTTTGCCCATGA 364 ETV1) GAACTAASouthern probe Chr 7 (5′ to NT_007819.16 13,734,028-13,734,003LNCAP_J2-r TCCCAGAAGAGATG 365 ETV1) ATATGAGGTGTC Southern probe Chr 7(5′ to NT_007819.16 13,737,858-13,737,881 LNCAP_K-f TCAGTCCCATCTCCC 366ETV1) CCTAAACCA Southern probe Chr 7 (5′ to NT_007819.1613,739,203-13,739,180 LNCAP_K-r CACCATTCTCACCCG 367 ETV1) ACCACATTGSouthern probe Chr 7 (5′ to NT_007819.16 13,743,031-13,743,060 LNCAP_L-fTGTAAACTGCAATGA 368 ETV1) AAAGAAAAGAAAAAG Southern probe Chr 7 (5′ toNT_007819.16 13,744,016-13,743,987 LNCAP_L-r CAAGAGATGGGAGA 369 ETV1)GGAAGAATGAATAATA Southern probe Chr 7 (5′ to NT_007819.1613,746,316-13,746,343 LNCAP_M-f CTATCTAGTCCCTTA 370 ETV1) CGCTTTCCCTGTGSouthern probe Chr 7 (5′ to NT_007819.16 13,747,138-13,747,116 LNCAP_M-rCATTAGCATTTGGCC 371 ETV1) TTTGGTCA Southern probe Chr 7 (5′ toNT_007819.16 13,754,560-13,754,584 LNCAP_N1-f TGCCTCCCATAAGT 372 ETV1)CACCAATCTC Southern probe Chr 7 (5′ to NT_007819.1613,755,734-13,755,705 LNCAP_N1-r CCTGTATTCTAACCC 373 ETV1)TGGACTTCTCATCAA Southern probe Chr 7 (5′ to NT_007819.1613,756,219-13,756,246 LNCAP_N2-f CTTGTTTATTGGCCT 374 ETV1) AGTCCTTTGTGCTSouthern probe Chr 7 (5′ to NT_007819.16 13,757,204-13,757,179LNCAP_N2-r GCTTTGTGGGTAGTC 375 ETV1) CTGTCTGAGTG Southern probe Chr 7(5′ to NT_007819.16 13,760,623-13,760,642 LNCAP_O1-f GGCCCATCCCGGTTT 376ETV1) GCTAA Southern probe Chr 7 (5′ to NT_007819.1613,761,824-13,761,798 LNCAP_O1-r GTTTCCCCACCACTT 377 ETV1) CCTTTCTATGTCSouthern probe Chr 7 (5′ to NT_007819.16 13,764,545-13,764,569LNCAP_O2-f GCACAAGACATACA 378 ETV1) CGCAGATACAC Southern probe Chr 7(5′ to NT_007819.16 13,765,703-13,765,678 LNCAP_O2-r AACGCTGGACTATG 379ETV1) GAACTTTACCTG Southern probe Chr 7 (5′ to NT_007819.1613,767,646-13,767,674 LNCAP_P1-f TCCTCTCATTCATTTT 380 ETV1)GCATTCGTGTTAG Southern probe Chr 7 (5′ to NT_007819.1613,768,845-13,768,819 LNCAP_P1-r GGCTTTGAGGGATTA 381 ETV1) CTGGGTTGTTCTSouthern probe Chr 7 (5′ to NT_007819.16 13,770,885-13,770,905LNCAP_P2-f GCAGGGCAAAGAAG 382 ETV1) CAGTAGG Southern probe Chr 7 (5′ toNT_007819.16 13,772,453-13,772,428 LNCAP_P2-r GGATCCCAATTTAGT 383 ETV1)TTCAAGTTACG Southern probe Chr 7 (5′ to NT_007819.1613,774,561-13,774,586 LNCAP_Q-f ATGTGCTGGCTAGAT 384 ETV1) TGGACTGAAAASouthern probe Chr 7 (5′ to NT_007819.16 13,775,272-13,775,244 LNCAP_Q-rCAATAAAGCTGGAG 385 ETV1) GGGTGATAAATAAAT Inverse PCR Chr 7 (Probe A)NT_007819.16 13,685,817-13,685,793 Inverse A1 TTAGAAGGAGACAA 386TCTTATTCCAG Inverse PCR Chr 7 (Probe A) NT_007819.1613685,657-13,685,634 Inverse A2 CTCTTAAAGAGATGA 387 AGCAGGGAG InversePCR Chr 7 (Probe A) NT_007819.16 13,685,626-13,685,603 Inverse A3TTGGCTAGATACAGG 388 GTGAATATT Inverse PCR Chr 7 (Probe A) NT_007819.1613,685,833-13,685,856 Inverse B1 TGAATTCATGTGTGT 389 AGCTGAGCC InversePCR Chr 7 (Probe A) NT_007819.16 13,685,860-13,685,883 Inverse B2TGACAGCGGGAATA 390 AAGTACATGC Inverse PCR Chr 7 (Probe A) NT_007819.1613,686,095-13,686,118 Inverse B3 GTTGGGAGGTTTACT 391 TGCCAATTA Genomicfusion Chr 7 (5′ to NT_007819.16 13,686,808-13,686,832 Genomic fusion-fACATTTTGCTAACCC 392 ETV1) CTCTTCTATCT Genomic fusion Chr 14 NT_026437.1118,985,248-18,985,272 Genomic fusion-r TCAACCTCAAAAATA 393 (MIPOL1)AATGGCATCT RWPE-ETV1 SERPINE1 NM_000602.1 1181-1200 SERPINE1-fGCATGGCCCCCGAG 394 GAGAT RWPE-ETV1 SERPINE1 NM_000602.1 1270-1248SERPINE1-r CTTGGCCCATGAAAA 395 GGACTGTT RWPE-ETV1 TGFBI NM_000358.11506-1528 TGFBI-f AGGTACGGGACCCT 396 GTTCACGAT RWPE-ETV1 TGFBINM_000358.1 1605-1580 TGFBI-r CTACCAGCATGCTAA 397 AGCGATTGTCT RWPE-ETV1IGFBP3 NM_000598.4 738-762 IGFBP3-f CGAGTCCAAGCGGG 398 AGACAGAATARWPE-ETV1 IGFBP3 NM_000598.4 837-814 IGFBP3-r TACACCCCTGGGACT 399CAGCACATT RWPE-ETV1 MMP3 NM_002422.3 1055-1080 MMP3-f TTCATTTTGGCCATC400 TCTTCCTTCAG RWPE-ETV1 MMP3 NM_002422.3 1181-1155 MMP3-rTATCCAGCTCGTACC 401 TCATTTCCTCT RWPE-ETV1 SPOCK1 NM_004598.3 829-848SPOCK1-f GCCCACCAGCTCCAA 402 CACAG RWPE-ETV1 SPOCK1 NM_004598.3 951-928SPOCK1-r GAAGGGTCAAGCAG 403 GAGGTCATAG RWPE-ETV1 BCL2 NM_000633.21014-1039 BCL2-f CCCTGTGGATGACTG 404 AGTACCTGAAC RWPE-ETV1 BCL2NM_000633.2 1084-1064 BCL2-r GGCATCCCAGCCTCC 405 GTTATC RWPE-ETV1 MMP14NM_004995.2 1036-1059 MMP14-f AATTTTGTGCTGCCC 406 GATGATGAC RWPE-ETV1MMP14 NM_004995.2 1151-1129 MMP14-r GGAACAGAAGGCCG 407 GGAGGTAGTRWPE-ETV1 MMP2 NM_004530.2 953-974 MMP2-f GAAGGCCAAGTGGT 408 CCGTGTGARWPE-ETV1 MMP2 NM_004530.2 1044-1019 MMP2-r CAGCTGTTGTACTCC 409TTGCCATTGAA RWPE-ETV1 ADAM19 NM_023038.2 2146-2165 ADAM19-fGCCTATGCCCCCTGA 410 GAGTG RWPE-ETV1 ADAM19 NM_023038.3 2271-2245ADAM19-r GCTTGAGTTGGCCTA 411 GTTTGTTGTTC RWPE-ETV1 MMP9 NM_004994.21181-1201 MMP9-f TGCCCGGACCAAGG 412 ATACAGT RWPE-ETV1 MMP9 NM_004994.21239-1221 MMP9-r AGCGCGTGGCCGAA 413 CTCAT RWPE-ETV1 PLAU NM_002658.21169-1194 PLAU-f TACGGCTCTGAAGTC 414 ACCACCAAAAT RWPE-ETV1 PLAUNM_002658.2 1308-1286 PLAU-r CCCCAGCTCACAATT 415 CCAGTCAA

TABLE 16 Probe # Gene/Region Localization Probe 1 ETV1 3′ RP11-124L22 2ETV1 5′ RP11-703A4 3 Chr 14q13.3-14q21.1 C RP11-945C4 4 Chr14q13.3-14q21.1 T RP11-107E23 5 HNRPA2B1 3′ 3′ RP11-11F13 6 HNRPA2B1 5′5′ RP11-379M24 7 HERV-K_22q11.23 5′ 5′ RP11-61N10 8 HERV-K_22q11.23 3′3′ RP11-71G19 9 SLC45A3 3′ RP11-249h15 10 SLC45A3 5′ RP11-131E5 11C15ORF21 5′ RP11-474E1 12 C15ORF21 3′ RP11-626F7 13 14q32 3′ RP11-483K1314 ETV1 5′ RP11-313C20 15 Chr 7 centromere C CEP 7 16 Chr 7p telomere TTelVysion 7p 17 TMPRSS2 5′ RP11-35C4

TABLE 17 International Genomics Consortium's expO Lynx TherapeuticsHuman Tissue dataset (Bittner_Multi-cancer at oncomine) MPSS Dataset(GSE1747) # Cancer Type n # Normal Tissue Type 1 Bladder PapillaryCarcinoma 4 1 adrenal gland 2 Bladder Transitional cell carcinoma 10 2Bladder 3 Breast Ductal Carcinoma 95 3 bone marrow 4 Cervix SquamousCell Carcinoma 10 4 brain - amygdala 5 Colon Adenocarcinoma 104 5brain - caudate nucleus 6 Metastatic Colon Carcinoma 16 6 brain -cerebellum 7 Mucinous Colon Carcinoma 12 7 brain - corpus callosum 8Endometrial Adenocarcinoma 5 8 fetal brain 9 Endometrial EndometrioidCarcinoma 45 9 brain - hypothalmus 10 Endometrial Mixed Mullerian tumor6 10 brain - thalamus 11 Metastatic Endometrial Carcinoma 7 11 Monocytes12 Soft Tissue Sarcoma 13 12 peripheral blood lymphocytes 13 Clear cellrenal carcinoma 78 13 Heart 14 Papillary Renal Cell Carcinoma 6 14Kidney 15 Lung Adenocarcinoma 19 15 Lung 16 Bronchioloalveolar carcinoma7 16 mammary gland 17 Squamous Cell Lung Carcinoma 17 17 Pancreas 18Ovarian Adenocarcinoma 20 18 pituitary gland 19 Ovarian EndometrioidCarcinoma 13 19 Placenta 20 Metastatic Ovarian Carcinoma 36 20 Retina 21Ovarian Mucinous Carcinoma 4 21 salivary gland 22 Ovarian PapillaryCarcinoma 38 22 small intestine 23 Pancreatic Ductal Carcinoma 3 23spinal cord 24 Rectosigmoid Adenocarcinoma 15 24 Spleen 25 RectalAdenocarcinoma 13 25 Stomach 26 Renal Pelvis Transitional cell carcinoma4 26 Testis 27 Metastatic Melanoma 5 27 Thymus 28 Papillary ThyroidCarcinoma 10 28 Thyroid Prostate Adenocarcinoma 15 29 Trachea 30 colontransversum 31 Uterus Prostate

B. Results Identification of Novel 5′ ETS Fusion Partners in ProstateCancer

By qPCR, two cohorts of prostate tissue samples were screened for ERGand ETV1 expression to identify cases with outlier-expression of ETV1.As shown in FIG. 38 a, across the two cohorts, 26 and 3 of 54 localizedprostate cancer samples showed ERG (48%) and ETV1 (5.5%)outlier-expression, respectively. Additionally, two hormone-refractorymetastatic prostate cancer samples, MET26 and MET23, showed ETV1outlier-expression. By qPCR, 25 of the 26 localized samples (96%) withoutlier-expression of ERG expressed TMPRSS2:ERG fusion transcripts.Besides MET26, no samples, including the 4 ETV1 outliers(PCa_ETV1_(—)1-3 and MET23), expressed TMPRSS2:ETV1 fusion transcripts.

To characterize the ETV1 transcript structure in these cases, 5′ RNAligase mediated rapid amplification of cDNA ends (RLM-RACE) wasperformed. Rather than 5′ exons from TMPRSS2 as in MET26, all foursamples contained unique 5′ sequences (FIG. 38 b). In PCa_ETV1_(—)1,exons 1-4 of ETV1 were replaced with two exons from 22q11.23 withhomology to the 5′ long terminal repeat (LTR) and gag sequence of humanendogenous retrovirus family K (hereafter referred to asHERV-K_(—)22q11.23). In PCa_ETV1_(—)2, exon 1 of ETV1 was replaced withexon 1 from HNRPA2B1 (7p15), while in PCa_ETV1_(—)3, exons 1-4 of ETV1were replaced with additional upstream sequence and exon 1 of SLC45A3(1q32). In MET23, exons 1-5 of ETV1 were replaced with exons 1 and 2from C15ORF21 (15q21) (FIG. 1 b). The exclusive expression of thesefusion transcripts was confirmed by qPCR and fusion at the genomic levelin these cases by FISH (FIGS. 38 c, 39 & 40). In PCa_ETV1_(—)2, FISHdemonstrated deletion of the probes 3′ to HNRPA2B1 and 5′ to ETV1, withcorresponding fusion of the probes 5′ to HNRPA2B1 and 3′ to ETV1,consistent with an intrachromosomal deletion between HNRP2A2B1 and ETV1,which are oriented head to tail ˜13 MB apart on 7p (FIGS. 38 c & 40).Sequences of gene fusions are shown in FIG. 51.

Distinct Functional Classes of 5′ Fusion Partners

HERV-K_(—)22q11.23:ETV1, SLC45A3:ETV1 and C15ORF21:ETV1 fusions containno predicted translated sequences from the 5′ partner, and the HNRPA2B1sequence in the HNRPA2B1:ETV1 fusion would contribute only two residuesto a fusion protein. Thus, as the promoter elements of the 5′ partnerslikely drive the aberrant ETV1 expression in these cases, the tissuespecificity and androgen regulation of these genes was characterized. Toexamine the tissue specificity of SLC45A3, C15ORF21 and HNRPA2B1, theInternational Genomics Consortium's expO dataset, consisting ofexpression profiles from 630 tumors of 29 distinct types, was searchedusing the Oncomine database (Rhodes et al., Neoplasia 9, 166-80 (2007)).Similar to TMPRSS2, SLC45A3 showed marked over-expression in prostatecancer (median=2.45 standard deviations above the median per array)compared to all other tumor types (median=0.33, P=2.4E-7). C15ORF21showed similar over-expression in prostate cancer (median=2.06 vs.−0.12, P=3.4E-6). By contrast, HNRPA2B1 showed high expression inprostate and other tumor types (median=2.36 vs. 2.41, P>0.05) (FIG. 38d). Although HERV-K_(—)22q11.23 is not monitored by the DNA microarraysused in the expO dataset, it is measured unambiguously by massivelyparallel signature sequencing (MPSS), as described by Stauffer et al(Cancer Immun 4, 2 (2004)). Thus, the expression of HERV-K_(—)22q11.23was searched in the Lynx Therapeutics MPSS dataset containing profilesfrom 32 normal tissue types (Jongeneel et al., Genome Res 15, 1007-14(2005)), which showed that HERV-K_(—)22q11.23 was expressed at thehighest levels in normal prostate tissue (94 transcripts per million)compared to the 31 other normal tissues (median=9 transcripts permillion) (FIG. 38 d).

By qPCR, the endogenous expression of SLC45A3 (21.6 fold, P=6.5E-4) andHERV-K_(—)22q11.23 (7.8 fold, P=2.4E-4) in the LNCaP prostate cancercell line are strongly increased by the synthetic androgen R1881,similar to TMPRSS2 (14.8 fold, P=9.95E-7). Conversely, the expression ofC15ORF21 is significantly decreased (1.9 fold, P=0.0012) by R1881stimulation. Lastly, the expression of HNRPA2B1 is not significantlychanged by androgen stimulation (1.17 fold, P=0.29)) (FIG. 38 e).

ETV1 Induces Invasion in Benign Prostate Cells

As the 5′ partners do not contribute coding sequence to the ETV1transcript, the common result of the different classes of ETV1rearrangements in clinical samples and prostate cancer cell lines isaberrant over-expression of truncated ETV1. Thus, this event wasrecapitulated in vitro and in vivo to determine the role of aberrant ETSfamily member expression in prostate cancer. Adenoviral and lentiviralconstructs were designed to over-express ETV1 as expressed in the indexTMPRSS2:ETV1 fusion positive case, MET26 (beginning at exon 4 throughthe reported stop codon of ETV1) (FIG. 41 a). The benign immortalizedprostate epithelial cell line RWPE was infected with lentivirusexpressing ETV1, selected for stable RWPE-ETV1 cells, and transientlyover-expressed ETV1 in the primary benign prostate epithelial cell linePrEC by infection with adenovirus expressing ETV1. In both RWPE and PrECcells, over-expression of ETV1 had no detectable effect on proliferation(FIG. 44 a-b), and cell cycle analysis showed no difference in thepercentage of RWPE-ETV1 and RWPE-GUS cells in S phase (FIG. 44 c).Additionally, soft agar transformation assays showed that ETV1over-expression was not sufficient to transform RWPE cells (FIG. 44 d).

ETV1 over-expression markedly increased invasion in both RWPE (3.4 fold,P=0.0005) and PrEC (6.3 fold, P=0.0006) (FIG. 41 b-c). Additionally,ETV1 knockdown in LNCaP using either siRNA or shRNA designed againstdifferent sequences significantly inhibited invasion (FIG. 41 d-e & FIG.45), consistent with previous work (Cai et al., Mol Endocrinol (2007)).These results demonstrate that ETV1 over-expression induces invasion, animportant oncogenic phenotype. To investigate the transcriptionalprogram regulated by stable ETV1 over-expression, RWPE-ETV1 cells wereprofiled and the expression signatures were analyzed against acompendium of biologically related concepts called the MolecularConcepts Map (MCM). The MCM is a resource to look for associationsbetween more than 20,000 biologically related gene sets bydisproportionate overlap (Tomlins et al., Nat Genet 39, 41-51 (2007)).As shown in FIG. 41 f, MCM analysis identified a network of molecularconcepts related to cell invasion that were enriched in our ETV1over-expressed signature, consistent with the phenotypic effectsdescribed above. By qPCR and immunoblotting, the over-expression ofmultiple genes previously implicated in invasion was confirmed (Laufs etal., Cell Cycle 5, 1760-71 (2006); Fingleton, Front Biosci 11, 479-91(2006)), including matrix metalloproteinases and members of theurokinase plasminogen activator pathway, in RWPE-ETV1 cells (FIGS. 41 g& 47). These results are consistent with a recent study demonstratingthat ETV1 mediates invasion in LNCaP cells through matrixmetalloproteinases (Cai et al., supra).

ETV1 Expression in the Mouse Prostate Induces mPIN

The effects of ETV1 over-expression in vivo was next investigated usingtransgenic mice expressing a FLAG-tagged, truncated version of ETV1under the control of the modified probasin promoter (ARR2Pb-ETV1) (FIG.41 a), which drives strong transgene expression exclusively in theprostate under androgen regulation (Ellwood-Yen et al., Cancer Cell 4,223-38 (2003)). This transgene is functionally analogous to theandrogen-induced gene fusions of ETV1 identified in human prostatecancer (i.e., TMPRSS2:ETV1, SLC45A3:ETV1, and HERV-K_(—)22q11.23:ETV1).Multiple ARR2Pb-ETV1 founders were obtained and expanded for phenotypicanalysis. By 12-14 weeks of age, 6 of 8 (75%) ARR2Pb-ETV1 transgenicmice developed mouse prostatic intraepithelial neoplasia (mPIN) (Table18 and FIG. 42). Consistent with the definition of mPIN, we observedfocal proliferative lesions contained within normal glands in theprostates of ARR2Pb-ETV1 mice (FIG. 42 a-f), exhibiting nuclear atypia,including stratification, hyperchromasia and macronucleoli. mPIN wasobserved in all three prostatic lobes (anterior, ventral anddorsolateral) of ARR2Pb-ETV1 mice, and most commonly in the ventral lobe(7/11, 63.6%) (Table 18). By immunohistochemistry, strong ETV1-FLAGexpression was observed exclusively in mPIN foci, and not benign glands,in ARR2Pb-ETV1 mice (FIG. 48), and qPCR confirmed that transgeneexpression was limited to the prostate.

All lesions were confirmed to be in situ by the presence of an intactfibromuscular layer, as demonstrated by contiguous smooth muscle actinstaining (FIG. 42 g-h). However, immunohistochemistry with the basalcell markers cytokeratin 5 and p63 demonstrated loss of thecircumferential basal epithelial layer in ARR2Pb-ETV1 mPIN compared tobenign glands (FIG. 42. i-l), indicating disruption of the basal celllayer. These results demonstrate that ETV1 induces a neoplasticphenotype in the mouse prostate and provides support of an oncogenicrole for ETS gene fusions in human prostate cancer.

TABLE 18 ARR2Pb-ETV1 Mouse Founder Age AP VP DLP Diagnosis Liver 1 28712 wk Normal Hyperplasia Hyperplasia Hyperplasia Normal 2 666 12 wkNormal Hyperplasia Normal Hyperplasia Normal 3 666 12 wk mPIN No tissuemPIN mPIN NA 4 666 12 wk Normal mPIN Adipose tissue mPIN Normal 5 28712-14 wk Normal mPIN Normal mPIN Normal 6 287 12-14 wk Normal NormalmPIN mPIN Normal 7 339 12-14 wk Normal mPIN Hyperplasia mPIN Normal 8339 12-14 wk Normal mPIN Hyperplasia mPIN Normal 9 287 19 wk NormalHyperplasia Hyperplasia Hyperplasia Normal 10 287 20 wk Normal mPINHyperplasia mPIN NA 11 287 33 wk Hyperplasia mPIN Hyperplasia mPINNormal 12 287 43 wk Normal mPIN mPIN mPIN Normal Total: 1/12 (8.3%) 7/11(63.6%) 3/12 (25%) 9/12 (75%)

Example 20 ETV5 Gene Fusions

This Example describes the identification of TMPRSS2:ETV5 andSLC45A3:ETV5 gene fusions. For detection of ETV5 outlier expression bySYBR Green QPCR, the following primer pair was used:

ETV5_Exon13-f: 5′-CCGAAGGCTTTGCTTACTAAGTTTCTGA-3′ (SEQ ID NO: 416)ETV5_Exon13-r: 5′-CACTGCCCTTGTTTGCCTGAATG-3′ (SEQ ID NO: 417)For identification of the TMPRSS2:ETV5 fusion from PCa_ETV5_(—)1, thefollowing primer for RLM-RACE was used:

ETV5_Exon6-r: 5′-AGCTCCCGTTTGATCTTGGTTGG-3′ (SEQ ID NO: 418)For identification of the SLC45A3:ETV5 fusion from PCa_ETV5_(—)2, thefollowing primer was used:

ETV5_Exon11-r: 5′-CATGGTAGGCTCCTGTTTGACTTTG-3′ (SEQ ID NO: 419)FIG. 52 shows the sequences of the above noted fusions. ForTMPRSS2:ETV5, 3 splice variants were identified. The sequences for allhave been given. FIG. 53 shows the identification of two prostate cancer(PCa) cases as showing ETV5 outlier expression by QPCR. Panel B showsthe structure of the fusion transcripts for both cases, as determined byRACE.

Example 21 Additional ERG, ETV1 and ETV4 Gene Fusions

Recurrent gene fusions involving ETS transcription factors ERG, ETV1,ETV4 or ETV5 have been identified in 40-70% of prostate cancers. Acomprehensive fluorescence in situ hybridization (FISH) split probestrategy was utilized to interrogate all 27 ETS family members and theirfive known 5′ fusion partners in a cohort of 110 clinically localizedprostate cancer patients. Based on this comprehensive FISH screen, fournoteworthy observations were made. First, a large fraction of prostatecancers (44%) cannot be ascribed to an ETS gene fusion. Second, SLC45A3is a 5′ fusion partner of ERG; previously, TMPRSS2 was the onlydescribed 5′ partner of ERG. Third, two prostate-specific,androgen-induced genes, FLJ35294 and CANT1, are 5′ partners to ETV1 andETV4, respectively. Fourth, a ubiquitously expressed,androgen-insensitive gene, DDX5, is fused in frame with ETV4 leading tothe expression of a DDX5:ETV4 fusion protein.

A. Materials and Methods Study Population, Clinical Data, and TissueMicroarray (TMA) Construction

A TMA was constructed representing 110 clinically localized prostatecancer patients who underwent radical prostatectomy as a primary therapybetween 2004 and 2006 at the University of Michigan Hospital. Cores (0.6mm in diameter) were taken from each representative tumor focus andmorphology was confirmed by pathologists. Detailed clinical,pathological, and TMA data were maintained on a secure relationaldatabase as previously described (Mehra et al. Mod Pathol. 2007; 20(5):538-44., herein incorporated by reference in its entirety). Patientdemographics are shown in Table 19. This radical prostatectomy serieswas part of the University of Michigan Prostate Cancer SpecializedProgram of Research Excellence Tissue Core.

TABLE 19 Clinical and Pathological Demographics of Patients CountPercentage (%) Age ≦60 57 52  >60 53 48 Gleason score sum   <7 13 12  =7 92 84   >7 5 4 Tumor size (cm)   <1 17 16  ≧1 88 84 Pathology stageT2 83 79 T3a 18 17 T3b 1 1 T4 3 3 Surgical margin Negative 82 75Positive 28 25 Preoperative PSA (ng/ml)  ≦4 13 12 4-7 50 48   >7 42 40PSA recurrence No 100 95 Yes 5 5 PSA, prostate-specific antigen Note:Not all data is available for all 110 patients

Fluorescence In Situ Hybridization (FISH) and FISH-Based ScreeningStrategy

A previously validated FISH-based split probe strategy was utilized toinvestigate known and novel gene aberrations in prostate cancer (Tomlinset al. Nature 2007; 448 (7153):595-9; Tomlins et al. Cancer Res 2006; 66(7):3396-400; Tomlins et al. Science 2005; 310 (5748):644-8., hereinincorporated by reference in their entireties). Bacterial artificialchromosomes (BACs) (listed in Table 20) were obtained from the BACPACResource Center (Oakland, Calif.), and probes were prepared.

TABLE 20 FISH Primers Gene Location 5′ 3′  1 EHF 11p13 RP5-1135K18RP5-1002E13  2 ELF1 13q14.11 RP11-88N4 RP11-53F19  2* ELF1 13q14.11RP11-110I22 RP11-71A13  3 ELF2 4q31.1 RP11-22O8 RP11-375P1  3* ELF24q31.1 RP11-1062K20 RP11-375P1  4 ELF3 1q32.1 RP11-25B7 RP11-246J15  5ELF4 Xq25 RP5-875H3 RP4-753P9  5* ELF4 Xq25 RP3-438D16 RP3-426N21  6ELF5 11p13 RP5-1002E13 RP5-1135K18  7 ELK1 Xp11.23 RP1-54B20 RP1-306D1 8 ELK3 12q23.1 RP11-69E3 RP11-510I5  8* ELK3 12q23.1 RP11-256L6RP11-587I15  9 ELK4 1q32.1 RP11-1089F13 RP11-1143H2 10 ERF 19q13.2RP11-208I3 RP11-317E13 11 ERG 21q22.2 RP11-137J13 RP11-24A11 11* ERG21q22.2 RP11-95I21 RP11-476D17 12 ETS1 11q24.3 RP11-75P14 RP11-112M22 13ETS2 21q22 RP11-24A11 RP11-137J13 14 ETV1 7p21.2 RP11-703A4 RP11-124L2215 ETV2 19q13.12 RP11-32H17 RP11-92J4 16 ETV3 1q23.1 RP11-91G5RP11-1038N13 16* ETV3 1q23.1 RP11-1150H17 CTD-2601M14 17 ETV4 17q21.31RP11-436J4 RP11-100E5 18 ETV5 3q27.2 RP11-379C23 RP11-1144N13 19 ETV612p13.2 RP11-90N7 RP11-59H1 20 ETV7 6p21.31 RP3-431A14 RP1-179N16 21 FEV2q35 RP11-316O14 RP11-129D2 22 FLI1 11q24.3 RP11-112M22 RP11-75P14 23SPDEF 6p21.31 RP11-79J23 RP11-119C22 24 SPI1 11p11.2 RP11-56E13RP11-29O22 25 SPIB 19q13.33 RP11-510I16 RP11-26P14 26 SPIC 12q23.2RP11-426H24 RP11-938C1 26* SPIC 12q23.2 RP11-36D3 RP11-631K19 27 GABP21q21.3 RP11-1130I5 RP11-1125C13 28 TMPRSS2 21q22.3 RP11-35C4RP11-120C17 29 SLC45A3 1q32.1 RP11-1089F13 RP11-1143H2 30 C15ORF2115q21.1 RP11-474E1 RP11-626F7 31 HERV- 22q11.23 RP11-947A12 RP11-61P17K_22q11.23 32 HNRPA2B1 7p15.2 RP11-379M24 RP11-11F13 33 FLJ35294 17p13.1RP11-1099M24 RP11-55C13 34 CANT1 17q25.3 RP11-52K16 RP11-46K10 35 DDX517q24.1 RP11-81D7 RP11-315N9 *tight probes set for corresponding ETSfamily genesThe integrity and correct localization of all probes were verified byhybridization to metaphase spreads of normal peripheral lymphocytes.Slides were examined using an Imaging Z1 microscope (Carl Zeiss,Oberkochen, Germany). FISH signals were scored manually (100× oilimmersion) in morphologically intact and non-overlapping nuclei bypathologists, and a minimum of 50 cancer cells from each site wererecorded. Cancer sites with very weak or no signals were recorded asinsufficiently hybridized. Cases lacking tumor tissue in all three coreswere excluded.

A flowchart of FISH-based high-throughput screening strategy is shown(FIG. 54). For detection of ERG, ETV1, ETV4 and ETV5 rearrangements inwhich breakpoints have been characterized, BACs for split probes wereused (Helgeson et al. Cancer Res 2008 68 (1):73-80, herein incorporatedby reference in its entirety). For the remaining ETS family genes withunknown rearrangement status in prostate cancer, split probes flankingapproximately 1 Mb regions of interest were utilized for initialscreening on the prostate cancer TMA (FIGS. 54A, B). Subsequently, ETSgene-specific probes tightly flanking the gene of interest(approximately 200 Kb) were used to confirm ETS aberration on tissuesections from prostate cancer cases that were identified on initialscreening (FIG. 54C).

This TMA was further evaluated for rearrangements of all 5′partnersknown at the time this study was started by split probe FISH strategy.For cases rearranged for both ETS gene and known 5′ partners, apreviously validated fusion probe strategy was utilized to confirmpotential gene fusions (FIG. 54D). For cases with ETS gene rearrangementonly, frozen tissues were obtained to identify 5′ fusion partner by RNAligase mediated rapid amplification of cDNA ends (RLM-RACE) (FIG. 54D).

Cell Line Studies

LNCaP, an androgen sensitive prostate cancer cell line, was maintainedin RPMI with 10% FBS. For androgen stimulation experiments, cells wereplaced for two days in phenol red-free RPMI, supplemented with 5% ofcharcoal-treated FCS before being treated with 1% ethanol or 10 nM R1881for the following time points 0, 3, 12, 24, and 48 hours. Cells weretreated for 16 hours with androgen and crosslinked for chromatinimmunoprecipitation (ChIP) analysis. Total RNA was isolated with Trizol(Invitrogen, Carlsbad, Calif.) according to the manufacturer'sinstructions.

Quantitative Real-Time Reverse Transcriptase PCR (Q-PCR)

Q-PCR was carried out according to standard protocols. Alloligonucleotide primers were synthesized by Integrated DNA Technologies(Coralville, Iowa) and are listed in Table 21.

TABLE 21 Oligonucleotide Primers SEQ Assay Gene Primer Sequence IDAndrogen/ Q-PCR F1 TTCGAGATGATGCCATTGAA 420 Expression FLJ35294Androgen/ Q-PCR R1 TCCTGAATTTTCTCCCATGC 421 Expression FLJ35294Androgen/ Q-PCR F1 ACGAGAACTGGGTGTCCAAC 423 Expression CANT1 Androgen/Q-PCR R1 ACTCCAGCAGGCAGACTCAT 424 Expression CANT1 Androgen/ Q-PCR F1TTCTAAATTTTTATGAAGCCAATTTC 425 Expression DDX5 Androgen/ Q-PCR R1CAGATCCAGTCTGTGCCACT 426 Expression DDX5 Androgen/ Q-PCR F1TGCACCACCAACTGCTTAGC 427 Expression GAPDH Androgen/ Q-PCR R1GGCATGGACTGTGGTCATGAG 428 Expression GAPDH RLM-RACE ETV1 ETV1_exon4-5_rCATGGACTGTGGGGTTCTTTCTTG 429 RLM-RACE ETV4 ETV4_exon4-rGAGCCACGTCTCCTGGAAGTGACT 430 RLM-RACE ETV4 ETV4_exon7-rGAAAGGGCTGTAGGGGCGACTGT 431 Expression ERG ERG_exon 5-6_fCGCAGAGTTATCGTGCCAGCAGA 432 Q-PCR Expression ERG ERG_exon5-6rCCATATTCTTTCACCGCCCACTC 433 Q-PCR Expression ETV1 ETV1_exon6-7fCTACCCCATGGACCACAGATTT 434 Q-PCR Expression ETV1 ETV1_exon6-7rCTTAAAGCCTTGTGGTGGGAAG 435 Q-PCR Expression ETV4 ETV4_exon11-fCTGGCCGGTTCTTCTGGATGC 436 Q-PCR Expression ETV4 ETV4_exon12-rCGGGCCGGGGAATGGAGT 437 Q-PCRSamples were normalized by the mRNA level of the housekeeping geneGAPDH. Androgen stimulation reactions were performed in triplicate, andall other reactions were performed in duplicate.RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE)

RLM-RACE was performed as to identify unknown 5′ partners of aberrantETS genes in prostate cancer. First-strand cDNA was amplified with genespecific reverse primers ETV1_exon4-5r, ETV4_exon7r and 5′ gene racerprimers (Invitrogen) using Platinum Taq High Fidelity enzyme(Invitrogen) following the touchdown PCR protocol according tomanufacturer's instructions. PCR amplification products were cloned intopCR4-TOPO TA vector (Invitrogen) and sequenced bidirectionally usingvector primers as described.

Chromatin Immunoprecipitation (ChIP) Analysis

ChIP experiment was carried out as previously described (Yu et al.,Cancer Cell 2007; 12 (5):419-31., herein incorporated by reference inits entirety) using 5 μg of anti-androgen receptor (AR) antibody(Upstate, Lake Placid, N.Y.) at 16 hours after R1881 or ethanoltreatment of hormone-deprived cells. The input whole-cell extract DNAand the ChIP-enriched DNA were amplified by ligation-mediated PCR, andsubjected to qPCR assessment of target promoters. ChIP enrichment wasevaluated as a percentage of input DNA.

Tissue-Specific Expression

To determine tissue-specific expression of 5′ fusion partners, theInternational Genomics Consortium's expO data set was interrogated,consisting of expression profiles from 630 tumours of 29 distinct types,using the Oncomine database.

Cloning and Expression of DDX5-ETV4

Full length DDX5-ETV4 fusion cDNA was cloned into Gateway cloningsystem's entry vector pENTR-D-TOPO (Invitrogen) using 5′RLM-RACE productfrom case no. 85 with 5′ primer containing a Kozak sequence-FLAGtag-start codon-DDX5 N-terminal nucleotide sequence(5′-CACC-ATG-GATTACAAGGATGACGACGATAAGTCGGGTTATTCGAGTGACCGAGACCGCGGC-3′;SEQ ID NO:438) and 3′ primer corresponding to the C-terminal nucleotidesof ETV4, until the stop codon (CTAGTAAGAGTAGCCACCCTTGGGGCCA; SEQ IDNO:439). Expression constructs were generated by recombination ofpENTR-D-Topo-DDX5-ETV4 with pcDNA3.2-DEST vector (Invitrogen), using LRClonase II (Invitrogen). Human embryonic kidney (HEK) 293 cells weretransiently transfected with pCDN3.2-FLAG-DDX5-ETV4 expression constructusing FuGENE6 transfection reagent (Roche). Lysates from the transfectedcells were resolved by SDS-PAGE, and transferred onto PolyvinylideneDifluoride membrane (GE Healthcare). The membrane was incubated for onehour in blocking buffer [Tris-buffered saline, 0.1% Tween (TBS-T), 5%nonfat dry milk] and incubated overnight at 4° C. with rabbit polyclonalantibody against FLAG tag at 1:1000 dilution (Cell SignalingTechnology). Following a wash with TBS-T, the blot was incubated withhorseradish peroxidase-conjugated secondary antibody and the signalsvisualized by enhanced chemiluminescence system as described by themanufacturer (GE Healthcare). The blot was re-probed with GAPDH (Abcam)for confirmation of equal loading. Prostate tissue lysates wereprocessed similarly for immunoblotting with ETV4 polyclonal antibody(Abnova).

B. Results

A comprehensive profile was generated of the rearrangement status of all27 ETS family genes and all five of the known 5′ fusion partners inprostate cancer using FISH split probe hybridizations on a TMA comprisedof 110 cases of clinically localized prostate cancers (FIG. 54).

A matrix representation of gene rearrangements for the 27 ETStranscription factors and the five 5′ fusion partners was generated(FIG. 55). ERG was the most commonly rearranged ETS gene in prostatecancer and TMPRSS2 was the most commonly rearranged 5′ fusion partner.In the cohort represented on this TMA, ERG was rearranged in 43% (43/99)of cases, of which 63% (27/43) were fused through deletion of its 5′ endto TMPRSS2, which is similar to previous reports (Perner et al. CancerRes 2006; 66(17):8337-41., herein incorporated by reference in itsentirety). Overall, 98% (42/43) of the ERG positive cases harboredTMPRSS2 as the 5′ fusion partner. One of the ERG positive cases wasnegative for TMPRSS2. Upon further inspection of our FISH screen (FIG.55), this case harbored rearrangement of the 5′ partner SLC45A3 (caseno. 102). The genomic fusion of SLC45A3:ERG were confirmed using afusion assay with probes 5′ to SLC45A3 and 3′ to ERG (FIG. 56), thusimplicating SLC45A3 as a novel 5′ fusion partner of ERG. Previous tothis finding, it was thought that ERG exclusively partnered with TMPRSS2possibly due to their co-localization on chromosome 21. SLC45A3 waspreviously identified to be the 5′ fusion partner of ETV1 and ETV5(Helgeson Cancer Res 2008; 68 (1):73-80., herein incorporated byreference in its entirety).

In contrast to ERG, ETV1 was rearranged in approximately 5% (5/99) ofpatients (case nos. 4, 51, 54, 72, 91, FIG. 55). ETV1 has been shown tobe fused to a number of genes including TMPRSS2, SLC45A3,HERVK_(—)22q11.23, C15ORF21 and HNRPA2B1. In the 110 cases representedin this study, case nos. 4 and 72 harbored rearrangements in ETV1 inaddition to rearrangements of SLC45A3 and HNRPA2B1, respectively. Fusionprobe FISH assays were utilized to confirm SLC45A3:ETV1 (a probe 5′ toSLC45A3 and 3′ to ETV1) and HNRPA2B1:ETV1 (a probe 5′ to HNRPA2B1 and 3′to ETV1) fusions in these two cases, respectively. Case no. 72 was thesecond case of HNRPA2B1:ETV1 reported in the literature, suggestingHNRPA2B1:ETV1 fusion is recurrent in prostate cancer. Case no. 4 whichharbored SLC45A3:ETV1 fusion was identified to be the same case asreported earlier (Tomlins et al. Nature 2007;448(7153):595-9). In theETV1 positive case nos. 51, 54, 91 no rearrangement in the known 5′partners were identified by FISH. In case no. 54, using RLM-RACE weidentified exons 1 to 4 of ETV1 to be replaced by 263 base pairs of the5′ sequence of a prostate EST gene FLJ35294 (17p13.1) (FIG. 57A).

ETV4 rearrangements were present in 5% (5/100) of patients in our cohort(case nos. 40, 46, 53, 64, 85, FIG. 55). Case nos. 64 and 85 hadrearrangement through translocation (split) while deletion of the probe5′ to ETV4 was identified in case nos. 40 and 46. In contrast, case no.53 revealed deletion of the probe 3′ to ETV4 (the significance of whichis unclear). Of note, case nos. 40 and 64 also showed an aberration forTMPRSS2 when screened by TMPRSS2 split probe. These two cases wereconfirmed to harbor TMPRSS2:ETV4 fusion using a fusion probe assay(employing probes 5′ to TMPRSS2 and 3′ to ETV4). In case no. 40, insteadof split signals, a 5′ end deletion was detected using probes 5′ and 3′to ETV4, suggesting that the TMPRSS2:ETV4 fusion in this case occurredthrough an unbalanced translocation. RLM-RACE further revealed that exon1 of ETV4 was replaced with exons 1-2 of TMPRSS2. Thus, this case isdifferent from the previously reported TMPRSS2:ETV4 fusion case where anovel upstream exon of TMPRSS2 was involved in the fusion (Tomlins etal. Cancer Res 2006; 66 (7):3396-400). ETV4 was rearranged in case nos.46 and 85, whereas no genetic aberration in the known 5′ partners wasobserved by FISH. RLM-RACE characterized the ETV4 transcript in thesetwo cases. In case no. 46, exon 5 of ETV4 was fused to exon 1a of theCANT1 gene located on 17q25.3, identical to the fusion sequence reportedbefore (Hermans et al. Cancer Res 2008; 68 (9):3094-8., hereinincorporated by reference in its entirety) (FIG. 57A). In case no. 85,another novel 5′ partner gene, DEAD (Asp-Glu-Ala-Asp) box polypeptide 5(DDX5) was identified by RLM-RACE (FIG. 57A). Sequence analysis ofDDX5-ETV4 revealed that the fusion transcript is comprised of exons 1-3of DDX5, fused in frame to exons 5-13 of ETV4 (FIG. 57A). Genomicfusions of FLJ35294:ETV1, CANT1:ETV4 and DDX5:ETV4 were confirmed byfusion probe FISH assay (FIG. 58).

No case with ETV5 rearrangement was identified in the present cohort.Among the remaining 23 ETS transcription factors, a small proportion ofprostate cancer cases revealed aberrant 5′ or 3′ deletions for ETS genesusing the FISH screening strategy (FIG. 55). Single cases of deletionswere found in the 5′ end of ETV3, ELF1 and SPIC and the 3′ end of ETV3,ELF2 and ELK3. Although FISH screening with a ˜1 Mb split probe approachyielded split signals for the ELK3 locus in case no. 12 and ELF4 locusin case no. 21, further FISH analysis by narrowing down the regionidentified the breakpoint location 220 Kb downstream of ELF4 gene and140 Kb upstream of ELK3 gene. Of note, BACs used to detect ELK4rearrangement were the same as utilized to detect SLC45A3 aberrationbecause of proximity (˜20 Kb) of the two genes. Split signals wereobserved in four cases (nos. 4, 39, 67 and 102). Of these, case no. 4and no. 102 were identified to 12 harbor SLC45A3:ETV1 and SLC45A3:ERGfusions, respectively. None of these cases harbored fusions between ELK4and SLC45A3. Thus, many of these singleton deletions are likelynon-specific, non-recurrent lesions.

Further, 110 patient TMA were screened for all of the known 5′ partnersof ETS aberrations in prostate cancer and found TMPRSS2 to be rearrangedin 47% of cases (48/102). SLC45A3 was rearranged in 2% (2/89), followedby HNRPA2B1 in 1% (1/99), and C15ORF2J in 1% (1/88) of cases. HERV-K_(—)22q11.23 rearrangement was not identified in this cohort. FISHanalysis revealed several cases with 5′ partner rearrangements withoutETS gene aberrations, indicating these ones may harbor non-ETS genefusion partners (FIG. 55).

The 5′ fusion partners of ETV1 and ETV4 identified in this study werecharacterized. By Q-PCR, ETV1 or ETV4 over-expression was confirmed incase nos. 54, 46, and 85 which harbored FLJ35294:ETV1, CANT1:ETV4 andDDX5:ETV4 fusions, respectively (FIG. 59A). As it is possible thataberrant ETS expression was driven by the regulatory elements ofFLJ35294, CANT1 and DDX5, their tissue specificities and androgenregulation in prostate cancer were examined. Similar to TMPRSS2,SLC45A3, FLJ35294 and CANT1 showed marked overexpression in prostatecancer compared to other cancer types using the International GenomicConsortium's expOdata set accessed in Oncomine (FIG. 57B,P=2.8×10-7(FLJ35294); P=6.8×10-7(CANT1)). By contrast, DDX5 exhibitedhigh expression in all tumor types (FIG. 57B) indicating that it mayhave a house-keeping function rather than prostate specificity.

By Q-PCR, expression of endogenous FLJ35294 (11.6-fold at 48 hours,P=0.0006) and CANT1 (3.8-fold at 24 hours, P=0.0014) were significantlyinduced by treatment with synthetic androgen R1881 in the LNCaP prostatecancer cell line (FIG. 59B). ChIP analysis demonstrated androgenreceptor (AR) occupancy of the promoter region in the FLJ35294 (9-foldenrichment, P=0.005) and CANT1 (40-fold enrichment, P=0.0014) genes(FIG. 59C). By contrast, the expression of DDX5 was not affected byR1881 stimulation (FIG. 59B) and AR was not recruited to the promoterregion of DDX5 (FIG. 59C). Therefore, the FLJ35294-ETV1 and CANT1-ETV4fusions can be categorized as class II gene fusions in prostate cancerwhich include rearrangements involving fusions from prostate-specificandrogen-induced 5′ partner genes; whereas DDX5-ETV4 may represent aclass IV gene fusion, in which non-tissue-specific promoter elementsdrive ETS gene expression.

FLJ35294-ETV1 and CANT1-ETV4 fusions contain no predicted translatedsequence (exons) from the 5′ partner. However, unlike almost allpreviously reported gene fusions in prostate cancer, the DDX5:ETV4fusion transcript codes for a putative fusion protein, with anN-terminal 102 amino acid stretch of DDX5 protein fused to 416C-terminal amino acids of ETV4 (FIG. 59D). A fusion protein is expressedby this chimeric transcript by cloning the full length cDNA from caseno. 85 into an expression vector encoding an inframe N-terminal FLAGepitope tag. Upon transient transfection into human embryonic kidney(HEK) 293 cells, the expression of the DDX5-ETV4 fusion protein wasdetected across independently derived clones (FIG. 59D). To assesswhether case no. 85 expresses the DDX5:ETV4 fusion protein, tissueextracts were exposed to immunoblot analysis using an antibody to theC-terminal end of ETV4 (FIG. 59D). Only the DDX5:ETV4 positive caseexpressed the aberrant 57 kDa fusion protein.

All publications, patents, patent applications and accession numbersmentioned in the above specification are herein incorporated byreference in their entirety. Although the invention has been describedin connection with specific embodiments, it should be understood thatthe invention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications and variations of thedescribed compositions and methods of the invention will be apparent tothose of ordinary skill in the art and are intended to be within thescope of the following claims.

1. A method for identifying prostate cancer in a patient comprising: (a)providing a sample from the patient; and (b) detecting the presence orabsence in the sample of a gene fusion having a 5′ portion from atranscriptional regulatory region of an SLC45A3 gene and a 3′ portionfrom an ERG gene, wherein detecting the presence in the sample of thegene fusion identifies prostate cancer in the patient.
 2. The method ofclaim 1, wherein the transcriptional regulatory region of the SLC45A3gene comprises a promoter region of the SLC45A3 gene.
 3. The method ofclaim 1, wherein step (b) comprises detecting chromosomal rearrangementsof genomic DNA having a 5′ DNA portion from the transcriptionalregulatory region of the SLC45A3 gene and a 3′ DNA portion from the ERGgene.
 4. The method of claim 1, wherein step (b) comprises detectingchimeric mRNA transcripts having a 5′ RNA portion transcribed from thetranscriptional regulatory region of the SLC45A3 gene and a 3′ RNAportion transcribed from the ERG gene.
 5. The method of claim 1, whereinthe sample is selected from the group consisting of tissue, blood,plasma, serum, urine, urine supernatant, urine cell pellet, semen,prostatic secretions and prostate cells.
 6. A method for identifyingprostate cancer in a patient comprising: (a) providing a sample from thepatient; and (b) detecting the presence or absence in the sample of agene fusion having a 5′ portion from a transcriptional regulatory regionof an FLJ35294 gene and a 3′ portion from an ETS family member gene,wherein detecting the presence in the sample of the gene fusion isidentifies prostate cancer in the patient.
 7. The method of claim 6,wherein the transcriptional regulatory region of the FLJ35294 genecomprises a promoter region of the FLJ35294 gene.
 8. The method of claim6, wherein the ETS family member gene is ETV1.
 9. The method of claim 6,wherein step (b) comprises detecting chromosomal rearrangements ofgenomic DNA having a 5′ DNA portion from the transcriptional regulatoryregion of the FLJ35294 gene and a 3′ DNA portion from the ETS familymember gene.
 10. The method of claim 6, wherein step (b) comprisesdetecting chimeric mRNA transcripts having a 5′ RNA portion transcribedfrom the transcriptional regulatory region of the FLJ35294 gene and a 3′RNA portion transcribed from the ETS family member gene.
 11. The methodof claim 6, wherein the sample is selected from the group consisting oftissue, blood, plasma, serum, urine, urine supernatant, urine cellpellet, semen, prostatic secretions and prostate cells.
 12. A method foridentifying prostate cancer in a patient comprising: (a) providing asample from the patient; and (b) detecting the presence or absence inthe sample of a gene fusion having a 5′ portion from a DDX5 gene and a3′ portion from an ETS family member gene, wherein detecting thepresence in the sample of the gene fusion identifies prostate cancer inthe patient.
 13. The method of claim 12, wherein the ETS family membergene is ETV4.
 14. The method of claim 12, wherein step (b) comprisesdetecting chromosomal rearrangements of genomic DNA having a 5′ DNAportion from the DDX5 gene and a 3′ DNA portion from the ETS familymember gene.
 15. The method of claim 12, wherein step (b) comprisesdetecting chimeric mRNA transcripts having a 5′ RNA portion transcribedfrom the DDX5 gene and a 3′ RNA portion transcribed from the ETS familymember gene.
 16. The method of claim 12, wherein step (b) comprisesdetecting a chimeric protein having an amino-terminal portion encoded bythe DDX5 gene and a carboxy-terminal portion encoded by the ETS familymember gene.
 17. The method of claim 12, wherein the sample is selectedfrom the group consisting of tissue, blood, plasma, serum, urine, urinesupernatant, urine cell pellet, semen, prostatic secretions and prostatecells.
 18. A composition comprising at least one of the following: (a)an oligonucleotide probe comprising a sequence that hybridizes to ajunction of a chimeric genomic DNA or chimeric mRNA in which a 5′portion of the chimeric genomic DNA or chimeric mRNA is from atranscriptional regulatory region of an SLC45A3 gene and a 3′ portion ofthe chimeric genomic DNA or chimeric mRNA is from an ERG gene; (b) afirst oligonucleotide probe comprising a sequence that hybridizes to a5′ portion of a chimeric genomic DNA or chimeric mRNA from atranscriptional regulatory region of an SLC45A3 gene and a secondoligonucleotide probe comprising a sequence that hybridizes to a 3′portion of the chimeric genomic DNA or chimeric mRNA from an ERG gene;(c) a first amplification oligonucleotide comprising a sequence thathybridizes to a 5′ portion of a chimeric genomic DNA or chimeric mRNAfrom a transcriptional regulatory region of an SLC45A3 gene and a secondamplification oligonucleotide comprising a sequence that hybridizes to a3′ portion of the chimeric genomic DNA or chimeric mRNA from an ERGgene; (d) an oligonucleotide probe comprising a sequence that hybridizesto a junction of a chimeric genomic DNA or chimeric mRNA in which a 5′portion of the chimeric genomic DNA or chimeric mRNA is from atranscriptional regulatory region of an FLJ35294 gene and a 3′ portionof the chimeric genomic DNA or chimeric mRNA is from an ETV1 gene; (e) afirst oligonucleotide probe comprising a sequence that hybridizes to a5′ portion of a chimeric genomic DNA or chimeric mRNA from atranscriptional regulatory region of an FLJ35294 gene and a secondoligonucleotide probe comprising a sequence that hybridizes to a 3′portion of the chimeric genomic DNA or chimeric mRNA from an ETV1 gene;(f) a first amplification oligonucleotide comprising a sequence thathybridizes to a 5′ portion of a chimeric genomic DNA or chimeric mRNAfrom a transcriptional regulatory region of an FLJ35294 gene and asecond amplification oligonucleotide comprising a sequence thathybridizes to a 3′ portion of the chimeric genomic DNA or chimeric mRNAfrom an ETV1 gene; (g) an oligonucleotide probe comprising a sequencethat hybridizes to a junction of a chimeric genomic DNA or chimeric mRNAin which a 5′ portion of the chimeric genomic DNA or chimeric mRNA isfrom a DDX5 gene and a 3′ portion of the chimeric genomic DNA orchimeric mRNA is from an ETV4 gene; (h) a first oligonucleotide probecomprising a sequence that hybridizes to a 5′ portion of a chimericgenomic DNA or chimeric mRNA from a DDX5 gene and a secondoligonucleotide probe comprising a sequence that hybridizes to a 3′portion of the chimeric genomic DNA or chimeric mRNA from an ETV4 gene;(i) a first amplification oligonucleotide comprising a sequence thathybridizes to a 5′ portion of a chimeric genomic DNA or chimeric mRNAfrom a DDX5 gene and a second amplification oligonucleotide comprising asequence that hybridizes to a 3′ portion of the chimeric genomic DNA orchimeric mRNA from an ETV4 gene; (j) an antibody to a chimeric proteinhaving an amino-terminal portion encoded by the DDX5 gene and acarboxy-terminal portion encoded by the ETV4 gene.